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Electrophoresis 1990,11, 562-568
T. Choli and B. Wittmann-Liebold
Theodora Choli Brigitte Wittmann-Liebold
Protein blotting followed by microsequencing The use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order toprevent N-terminal blockage ofthe proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment for in situ amino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer).
1 Introduction Blotting procedures in combination with microsequencing will continue to be important for the exploration of protein structure. Increased sensitivity in protein analysis has been achieved by systematically improving both the protein purification and sequencing techniques. Although high performance liquid chromatography (HPLC) is widely used for protein isolation [ 1-71 and permits sequence analysis of picomole quantities [SI, sometimes an efficient HPLC resolution for fully purified polypeptide chains cannot be achieved [6,71. Another alternative preparation technique, the electroelution of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or urea gels [8-15], cannot be recommended for polypeptides for subsequent Edman degradation or amino acid analysis, due to coelution of contaminants present in the electrophoresis system. An improvement is the electroblotting of proteins separated by PAGE [8, 17-20]. However, the membranes used for Western blotting 121-231 (commonly referred to as “protein blotting”) e. g., cellulose or nylon derivatives, are chemically unstable and therefore cannot be applied for Edman degradation and amino acid analysis in situ on the membrane. C)n the other hand, derivatized glass filters [16, 17, 201 or Polybrene-coated glass supports [ 161, and synthetic fibers, such as polyvinylidene difluoride (PVDF) [ 18,191 allow in situ NH,-terminal sequencing of proteins, amino acid analysis and determinationof internal sequences after protein blotting. The protein amounts needed for these techniques depend on (i) the type of membrane used, (ii) the hydrophilicity/hydrophobicity of the examined protein, and (iii) the applied sequencing strategy.
2 Blotting of proteins for sequencing 2.1 Prevention of N-terminal blockage PAGE, in combination with blotting to sequencer-stable supports has proven to be a powerful tool for the detection, separation and analysis of proteins. Proteins separated on the basis of their molecular masses [21,24,251 or by two-dimensional electrophoresis [26-321, based om charge differences followed by molecular mass separation in the second dimension, can be further characterized by blotting onto sequencerstable supports such as modified glass fibers (GF) or PVDF Correspondence: Dr. Brigitte Wittmann-Liebold, Max-Planck Institut fur Molekulare Genetik, Abteilung Wittmann, D-1000 Berlin 33 (Dahlem), Germany Abbreviations: PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid (3VCH Verlagsgesellschaft mbH D-6940 Weinheim, 1990
Immobilon (Millipore, Bedford, MA). The elucidation of the primary structure using the Edman chemistry needs a free amino-terminal group. Consequently, the first major consideration for proteins when subjected to electrophoresis, blotting and sequencing by the Edman method is the prevention of any N-terminal modification of the polypeptide chain. A considerable number of precautions should be routinely followed in the preparation of samples to be sequenced by this technique. 2-Mercaptoethanol or dithioerythritol are added to all buffers, used for the isolation and purification steps,in order to avoid formation of aggregates through disulfide bridge formation and the attack of the amino group by radicals, aldehydes or oxygen. T o protect the eluted proteins from possible oxidation, these mercaptans are also used during electrolution. Gel preruns, and the addition of thioglycolic acid (0.1 mM) to the running buffer [ 19, 33, 341 during preelectrophoresis have been reported to increase the initial yields and to prevent tryptophan and methionine from degradation [341.
2.2 Blotting from SDS-PAGE In 1973 Jovin [35-381 published a theory ofmultiphasic zone electrophoresis, making it possible to optimize the choice of buffer system in electrophoresis. According to Geisthard and Kruppa [ 391, amino-containing compounds in the electrophoresis buffer and some of the additives proposed in [381, e. g . thioglycolic acid, react at alkaline pH with residual acrylamide monomers, still present in the polyacrylamide gels after polymerization. Nucleophilic addition of ammonia, primary and secondary amines [40-421, and amino acids 143-441 to a, P-unsaturated carbonylic compounds is a wellknown chemical reaction. Accordingly, the residual acrylamide monomers react with the a-NH,-terminal and the &-NH,-groupsof lysines in proteins and peptides with resultant changes in electrophoretic mobility [391. Alkylation of proteins by acrylamide has also been described [45,461. Strategies to eliminate the accumulation of byproducts in the gel would be beneficial. A preelectrophoresis step and addition of thioglycolic acid to the running buffer would have three effects: (i) removal of charged impurities, (ii) reduction of peroxides and residual radicals, and (iii) scavenging of uncharged reactive species such as acrylamide monomer, free or incorporated acrolein, or other reactive carbonyl compounds. Unfortunately, a preelectrophoresis step in a discontinuous system may result in band diffusion and an impaired separation. Moos and co-workers [34l have suggested a modified, continuous Laemmli system, allowing a separation at pH 7.8 and consequently eliminating the acrylamide monomer reaction with the free a-amino groups of the proteins. This buffer system is recommended for blotting of protein mixtures with closely adjacent bands. A preelectrophoresis step of the nor01 73-0835/90/0707-0562 $3.50+.25/0
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Electroohoresis 1990,Il. 562-568
Figure I. Separation of human heart muscle proteins by one-dimensional (5 %- I 5 %)gradient SDS-PAGE.(A) Protein staining after preelectrophoresis and electrophoresis. (B) Stained protein blot on a PVDF membrane after preelectrophoresis and electrophoresis. ( C )Staining after electrophoresis without preelectrophoresis). Lane (1) marker proteins: (A) 200 kDa; (B) 97.4 kDa;(C) 68 kDa;(D)43 kDa;(E) 29 kDa;(F) 18.4 kDa; (G) 14.3 kDa; lanes (2)-(4) different amounts of heart muscle proteins: 20 pg total protein (left lane and 40 pg (right lane) each. Proteins were electrotransferred onto a PVDF membrane for 1 h using the semidry method with 1.8 mA/cmZ at 4 O C . Staining was done as described in 1331.
mal discontinuous Laemmli system [ 19,331 does not lead to drawbacks if the protein bands in the gel are well resolved. Thus, for optimal performance the SDS gels were prepared a day ahead to reach a high polymerization rate and, depending on the gel size (1 3 x 20 cm or 7 x 8 cm), they were preelectrophoresed at 4 "C overnight or for 2 h, respectively, in a buffer supplemented with 0.01 mM thioglycolic acid. SDS-PAGE of marker and heart muscle proteins, with or without a prerun, did not result in diffusion (Fig. 1) which, however, was observed for other proteins following preelectrophoresis (Fig. 2).
2.3 Blotting from urea gels The interaction of urea with proteins and polypeptides has been studiedextensively [47-5 11. Cyanate andcarbonateions may accumulate in aqueous urea solutions at neutral and alkaline conditions and are most pronounced at a pH > 4-5 [52,53]. The rate of reaction of cyanate with amino groups of proteins appears to involve the unprotonated amine and the unionized cyanic acid: R-NH,
Figure 2. SDS-PAGE (15 % discontinuous system) according to Laemmli 1241 (A) Without and (B) with preelectrophoresis overnight at 4 O C . Left lanes: 50 pg total protein of the ribosome from the archaebacterium Sulfolobus acidocaldurius:right lanes: 10 pg of ribosomal proteins ~ 1 210/ and their fragment (P. Henning, personal communication).
+ NH=C=O + H2O -+ R-NH-CO-NH2 + HZO
This is consistent with the difference in the reaction rates shown by a-amino ( ~ K A 8) = and E-amino groups ( ~ K A 10.7) = of proteins. At pH 7 a-amino groups react 100 times faster than the E-amino groups [551. Carbamylation is not aproblem at p H < 4 and pH > 10I26,561. For this reason ribosomal proteins of E . coli were separated in 6 M urea on CM-cellulose columns under acidic conditions which did not block the N termini of the proteins. The cyanate concentration must be kept low, otherwise the protein will be carbamylated. At low temperatures, the formation of the cyanate ions in urea solutions is extremely slow [52].The acidic ribosomal proteins of Hafobacterium marismortui (Hma-rib proteins) f2, which are only soluble in high molar urea sohtions (6-8 M), ~were maintained in a sequenceable form after solubilization at -20 "C 1191. In addition, the urea used for two-dimensional 5413
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T. Choli and B. Wittmann-Liebold
choice of support and transfer conditions is critical in protein blotting, which is anticipated to provide a true replica of the original gel pattern. The degree to which this is achieved depends mainly on the choice of transfer buffer, the equilibration conditions prior to the transfer and blotting equipment. The electrophoretic transfer of proteins from polyacrylamide gels to the sequencer-stable membranes is performed by two blotting techniques (i) the conventional tank system [22, 58, 591 and (ii) the semi-dry system 1601. For an efficient protein transfer, the entrapment of air bubbles must be avoided in both systems. This can be achieved by pressing the membrane sheet onto the gel surface with a glass rod or spatula. The semi-dry system affords the following advantages over the conventional buffer tank system: (i) The transfer time is drastically reduced and, consequently, the polypeptides are less exposed to oxidation processes. (ii) The filter papers wetted with the buffer are the only buffer reservoirs in the apparatus. (iii) The use of a discontinuous buffer system results in low Joule heat generation 1601. Hence the consumption of buffersin the semidry apparatus is strongly reduced in comparison to the tank version, typically to one tenth of the amount needed in thelatter.
2.5 Preequilibration
Figure 3. Blot onto two consecutive PVDF mernbranes of ribosomal pro teins from the 50s subunit of Bacillus stearothermophilus. Two-dimensional electrophoresis according to 1271; blotting conditions as in 1331. (A) First PVDF membrane; (B) second PVDF membrane.
electrophoresis was of high purity (ultrapure, BRL, Gaithersburg, MD) in order to avoid carbamylation effects. For other ribosomal proteins such as from Bacillus stearothermophilus or from E. coli (Fig. 3) the gel system employed was as describedin 126,271.Theproteinswereextractedfrom70S, 50S, or 30s subunits [571, except that all dialysis buffers contained 10 mM 2-mercaptoethanol. The final dialysis was against 2 % acetic acid with 10 mM 2-mercaptoethanol, which kept the ribosomal proteins from both organisms in a soluble state before lyophilization. The gels were predectrophoresed in the presence of thioglycolic acid for about 3h at 4 "C. After the proteins were electrophoretically transferred onto PVDF membranes [33],they were stained with Amido Black and the excised bands were directly sequenced using the pulse-liquid gas-phase sequencer (Applied Biosysterns, Foster City, C A).
2.4 Electrophoretic transfer The advantage of protein blotting onto membranes is that the transferred proteins are available for further characterization. The proteins are bound to the surface of the sequencer-stable membranes by hydrophobic or electrostatic interactions. The
Independent of the blotting system the gels have to be preequilibrated with the transfer buffer in order to prevent a possible change of buffer conductivity and gel swelling during the transfer, which is performed by applying a constant current. Since proteins are usually eluted from SDS-containing gels, the effect of SDS during transfer and protein immobilization has to be considered. SDS forms a complex with proteins yielding highly negatively charged molecules [6 1I. By using a single transfer buffer for all types of proteins, it is unlikely that all of them will quantitatively be transferred from SDS-PAGE gels, mainly because of the preferential loss of SDS from the gel. As long as SDS remains in the gel, the proteins are in a soluble form. However, owing to its small size and negative charge, SDS migrates out of the gel faster than most proteins, which are then trapped within the gel. Inclusion of SDS in the equilibration buffer considerably improves the transfer efficiency [621.According to I171 the SDS was displaced from the proteins by immersing the gels in 0.5 % v/v Nonidet P-40 for 10 min at room temperature. Most proteins possess two classes of SDS binding sites, i. e. strong binding sites, saturated at 0.4 g SDS/g protein and weaker binding sites saturated at 1.4 g SDS/g protein. A complete removal of SDS from its complexes with proteins is not easy to achieve 163-451. The preequilibration time should not be longer than 10- 15 min in order to avoid band diffusion or leaching of low molecular weight proteins from the gel.
2.6 Transfer buffers Different transfer buffers have been proposed. According to [ 171 the transfer from SDS gels onto chemically activated glass fiber sheets was efficient when 1 % acetic acid [661 and 0.5 % Nonidet P-40 were used. The transfer buffers employed for blotting onto PVDF membranes consist of 25 mM TrisHCI, pH 8.4, 0.02 % SDS and 0.5 % dithiothreitol 119, 331. SDS was added to the transfer buffer in order to improve the transfer of high molecular weight proteins which might otherwise be trapped within the gel. Binding ofthe transferred proteins to the PVDF membrane was not affected by the presence of the detergent (T. Choli, unpublished results) but
Electrophoresis 1 9 9 0 , I I . 562-56s
Protein blotting followed by microsequencing
SDS was found to interfere with protein binding to the Polybrene-coated glass fibers and to derivatized nitrocellulose membranes [ 16,621. However, also in the case of the PVDF membranes, the detergent (SDS) concentration in the transfer buffer should be low; otherwise, artifacts will appear during the first few cycles of automated Edman degradation, interfering with the assignment of amino acid residues 1671. In addition, SDS may decrease the pH of the coupling reaction with partial overlaps on degradation as well as slower drying times of the sample after the coupling and cleavage stages 1681.
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0.1 % Amido Black, in 50 % methanol for 5 min, and destainedin 30 % methanolfor 5-10minatroomtemperature.Staining of PVDF membranes with Coomassie Brilliant Blue or Amido Black leads to lower initial yields in the sequence compared with those of the unstained pieces (see Table 1) [331. After washing and detection, the protein bands are excised and either sequenced immediately or stored at -20 “C under nitrogen in sealed Eppendorf tubes.
3 Sequencing supports The use of methanol, commonly 10-20 %,in the transfer buffers seems to be another critical factor [ 16, 18, 20, 691. According to I 161, swelling of the polyacrylamide gel is avoided and precipitation of the SDS-Polybrene salt on the glass fiber is probably reduced. On the other hand, methanol contains aldehydes which block proteins by formation of Schiffs bases. We did not include methanol in our transfer buffer 19, 331 because no improvement in the transfer efficiency or proteinmembrane binding was noticed with our proteins. The discontinuous buffer used in our experiments has the following composition: the cathode buffer consists of0.04 ~6-amino-n-hexanoic acid, 0.025 M Tris-HC1,20 %v/v methanol, p H 9.4, and that ofthe anode buffer of0.3 MTris-HCl, 20 %v/vmethanol, pH 9.4, and that ofthe anode buffer of0.3 MTris-HCl, 20 % v/ v methanol, pH 10.4, in order to neutralize protons produced at the anode during electrophoresis.
3.1 Glass fiber activation/derivatization
Activated or derivatized glass fibers were the first membranes successfully employed for microsequencing of blotted proteins [ 16, 17, 201. They are activated by immersing in 100 % trifluoracetic acid (TFA) for 1-4 h, drying, and washing in methanol [17, 191. An additional activation step can be performed by immersing the sheets in potassium hydroxide saturated ethanol for 12 h, then in 3 N hydrochloric acid for 12h, or 0.1 %hydrogen fluoride in water for 15 rnin L 191. The activation is followed by derivatization using 3-amino-propyltriethoxysilane or 1,4-phenylene diisothiocyanate [ 171. Polybrene-treated glass fiber membranes are prepared by allowing a solution of 3 mg/mL of Polybrene in water to drip from a Pasteur pipette over the glass fiber sheet. The impregnated sheets are dried in air and are stored at -20 “C. Before use, excess Polybrene is washed out ( 5 rnin with 1 0 0 m L 2.7 Detection of transferred proteins bidistilled water) and the wet sheets are mounted for blotting. After transfer, the membranes are usually washed briefly by Recently, another type of derivatization of glass fiber sheets rinsing in bidistilled water 116, 19, 331,3 x for 10-15 min, to has been reported to show an excellent protein-binding caremove gel contaminants. Proteins blotted onto PVDF mem- pacity [201. According to these researchers the activation branes can be detected without staining as grayish areas on a step was performed by refluxing the sheets in T F A containwhitemembranebackgroundupondrying [16,19).Iflessthan ing 0.2 % poly(methyl-3,3,3-trifluoropropylsiloxane)for 10h 50 pmol of protein per cm2are transferred, they are not visible with gentle stirring. After reaction, the wet sheets were placed [161 and the proteins must be stained with Coomassie Bril- in a desiccator and TFA was removed under vacuum. The liant Blue, Amido Black, Ponceau S [701 or visualized with sheets were then rinsed with acetone toremove excess reagent. fluorescent dyes [ 161. Fluorescamine, reacting with primary Curing of the siloxane linkage was achieved by drying the amino groups to yield fluorescent derivatives I7 11, irreversibly sheets for 60 rnin at 150°C. These sheets are available as blocks a-NH, groups. According to [ 161 this undesirable “Glassy bond” filters (Biometra, Gottingen, FRG). reaction can be avoided by using dilute solutions of 1 mg/200 mL or less. Staining with Ponceau S [701 has the same sen3.2 PVDF membranes sitivity as Coomassie Brilliant Blue R-250 (250 to 500 ng protein), and can be completely removed from the protein by rinsP V D F membranes I181 are mechanically stable, solid-phase ing the stained membrane with water for additional 10-15 supports that bind proteins by hydrophobic interaction. They min. However, its influence on the initial yields of degradation have been used in immunoblotting applications as a substitute has not yet been studied. Most commonly the membranes are for nitrocellulose membranes [721. PVDF membranes are instained with either 0.1 % Coomassie Brilliant Blue R-250 in ert to most solvents (acetonitrile, TFA, ethyl acetate, trimeth50 % methanol, for 5-10 rnin at room temperature, or with ylamine and hexane, but not dimethylformamide), and can be used in automated sequencing. Due to its high hydrophobiciTable 1. Effect of the position of the protein blots in the cartridge block in initial sequencing yield@ ty this membrane should be immersed in 100 % methanol for about 20s [ 181 and equilibrated with the transfer buffer used Detection Arrangement Yield (Yo) for the electrotransfer before placement onto the gel. Coomassie Brilliant Bluebj Amido Blackh, Unstained Unstained
Protein facec) Protein face Protein face Random
24.1 Yo 15.0 % 40.0 % 21.8 %
a) P-Lactoglobulin samples (500 Pmol) were applied onto the gel and blotted after electrophoresis. Initial yields were determined from the NH,terminal residues. The values shown are the percentages ofthe amounts of the protein loaded onto the gel. The yields are average values from three experiments. From 1331 b) Weakly stained for 5 min c) The “protein face” arrangement is depicted in Fig. 4
3.3 Protein binding capacity of various activated and derivatized membranes The above-mentioned membranes have been tested for their binding capacity for standard proteins, i. e. Bst ribosomal proteins (mostly very basic), and Hma (generally acidic) as described in [ 191. The binding capacity has been determined as the 18 amount of protein bound to 1 cm disks of the membranes. The P V D F membranes were found to pos-
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T. Choli and B. Wittmann-Liebold
Table 2 Protein binding capacity of sequencer-stable membranes Support
Capacitya)
Activated glass fibers TFA Hydrogen fluoride Hydrochloric acid Potasssium hydroxyde Derivatized supports (glass fibers) Polybrene Aminopropyl Quaternary ammonium Diazotized Diisothiocyanate Polybase-coated glass fibers Polyvinylidene difluoride
5-8 5-8 5-8 5-8 25-35 4-6 4-6 4-6 20-30 30-40
a) Values are given in kg protein/l cm disk of support. The values for activated supports are for proteins blotted from acetic acid-urea gels; the values for the other supports are for proteins blotted from SDS-PAGE gels. From [ 191
sess a higher binding capacity than the derivatized glass membranes, similar to that of Polybrene-treated glass fibers (Table 2).
3.4 Extraction of proteins blotted onto sequencer-stable supports Conditions for extracting proteins blotted onto PVDF or Polybrene-coated glass fiber disks have been reported by several workers [73,741. The efficiency of elution depends on several factors, e. g., hydrophobicity, size of molecule and type of membrane used. It should be noted that once the sample is exposed to sequencing conditions, i. e. after 3 cycles of Edman degradation, the extraction is virtually impossible [721. Proteins blotted onto Polybrene-coated glass fiber sheets can be eluted with 80 % HCOOH [73].. For the extraction of proteins blotted onto PVDF membranes 70 % isopropanol in 5 % TFA was the best volatile solvent combination for adequate extraction (721 with about 60 ‘%proteinrecovery, as assessed by sequencing and determination of initial yields. The proteins were excised and suspended in 0.3 mL of solvent, vortexed and incubated at room temperature for 60 min. The membrane was then removed for sequence analysis, and the solvent was evaporated in a Speed Vac centrifuge and dis-
I+
solved in 30 FL 0.1 % TFA for sequence analysis. Buffer solutions containing SDS and Triton X-100 have also been proposed 1741.Thus, proteins were completely eluted from PVDF membranes at room temperature by short incubation in 50 mM Tris-HC1, p H 9.0, containing 2 % SDS and 1 % Triton X- 100 and the efficiency of elution was practically independent of the molecular weight of the proteins. However, SDS or Triton should be removed from the extracted proteins prior to microsequencing, which is accompanied by further sample loss. Therefore buffers containing detergents or other nonvolatile components should be avoided if the sample is to be used for microsequencing or amino acid analysis.
4 Microsequence analysis 4.1 Microsequence analysis of proteins immobilized on PVDF membranesin the Applied Biosystems sequencer The arrangement in the reactor of pieces of PVDF-blotted protein is an important factor in sequence analysis (Fig. 4) t 19, 331. The membrane surface onto which the protein is blotted (showing the highest intensity) is placed in the upper cartridge in upside-down position, followed by the second PVDF membrane. Both PVDF membranes are kept in position by covering them with a TFA-treated glass fiber, pretreated with 2 mg Polybrene [75,761 andprecycledin anormal sequencing program prior to blotting. The upper cartridge is then turned over and screwed onto the lower cartridge part so that the proteins are on top of the filters. At the R, delivery and wash stages, the blotted proteins are eluted from the PVDF membrane, transferred to and trapped on the pretreated glass filter. With this technique the blotted proteins are degraded mainly in the glass filter employing a normal degradation program. Incomplete coupling due to insufficient penetration of the aqueous base into the PVDF membrane is thereby prevented. According to [771, the glass fiber filter should be removed from the cartridge, otherwise substances may be adsorbed to it instead of to the hydrophobic membrane. Table 1 shows the yield differences between blotted, unstained P-lactoglobulin samples, arranged in the cartridge either with the proteins on top (as described above) or randomly arranged. The initial yields of 40 % in the first case, and 2 1.8 %
a
/
-
1 Membrane
/
Figure 4 . (A) Electrotransfer from an SDSgel onto PVDF membranes. The arrow indicates the blotting direction and (a) shows the membrane surface on which most of the protein was detected. (B) Arrangement of the PVDF pieces in the cartridge of the sequencer. Number 1 and 2 are the first and second PVDF blot, respectively; GF glass fiber disk, pretreated with TFA and precycled with Polybrene.
Electrophoresis 1990,11, 562-568
in the second, demonstrate that the correct sequenceofPVDF membranes is important. Repetitive yields from proteins adsorbed on PVDF membranes were 1-2 % lower compared to that obtained for the same proteins adsorbed to Polybrenetreated fiber filters [ 181. In this case the PVDF membrane was centered onto the Teflon seal without Polybrene-treated glass fibers and placed directly in the cartridge block of the sequencer. The lower initial yields could result from a combination of incomplete cleavage, low coupling, or washing out of peptides. PVDF membranes have a 0.45 pm pore size and are thinner than a glass fiber disk, thus higher liquid volumes are delivered due to the lower back pressure of this membrane. Therefore, too much acid is delivered in the normal degradation program, causing protein loss. The solvent wash steps in the sequencing programs are excessively long for sequencing proteins absorbed onto these membranes. To circumvent these problems and to use the normal sequencing program we prefer to cut the PVDF blot into pieces (a maximum of 3-5 pieces of 4 mm’) which then are placed on top of a precycled Polybrene-treated glass fiber filter. Another efficient approach uses pretreatment of the PVDF membrane with Polybrene, which gave higher initial yields in comparison to a similar electrotransfer on derivatized glass fiber supports 1721. 4.2 Microsequence analysis of proteins immobilized on PVDF membranes in the Knauer sequencer
Protein blotting followed by microsequencing
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quencing yields are caused by incomplete cleavage in the gel and the formation of several peptides derived from the same protein region. Newer approaches to obtain internal sequence information are to fragment the blotted proteins in situ by proceduresgivenin [33,73,8 11. Inall thesecases,independent of the membrane type used, the membrane pieces (after the transfer) have to be pretreated with polyvinylpyrrolidone (PVP-40) in order to prevent adsorption of the enzyme to the blot-support during digestion. After pretreatment, excess PVP-40 should be removed by extensive washing with water because this chemical strongly absorbs in the UV region. PVDF membranes for blotting were treated with 0.2 % PVP40 in 100 % methanol, followed by incubation in 0.5 % PVP40 in 100 mM acetic acid. Digestion proceeded for 20 h [33], 24 h [731 or overnight (811 at a 1 :20 protein-to-enzyme ratio. For digestion a 100 mM N-methylmorpholine acetate buffer, pH 8.0, is preferable to a bicarbonate buffer or 100 mM TrisHCl, pH 8.5. Carbonate buffers cannot be recommended because they block N-terminal amino groups by Schiff-base formation which becomes a serious disadvantage for blotting of picomole protein quantities. The use of organic solvents 133, 73, 811 in the digestion solution is not always necessary and depends on the protein character and the applied membrane. In case of PVDF blots the presence of 20 % methanol or acetonitrile in the digestion buffer is recommended but is not necessary if an N-methylmorpholine buffer is used [331. For chemical cleavages, e. g. by CNBr treatment of small proteins, the application of PVP-40 can be omitted, while for the fragmentation of bigger molecules its addition is necessary [331.
The Berlin sequencer (Dr. H. Knauer GmbH, Berlin, FRG) has a flow-through reactor [ 781 which can easily accomodate PVDF or glass filter supports. Usually, the oval filters of 6 x 12 mm in diameter are placed into the glass inlet of the reactor followed by protein loading in 5- 10 1 L of 50 % TFA in water. Careful reading of the English version of the manuscript by Blotted protein filters of the same size can be inserted directly Dr. A d a m Inglis is greatly acknowledged. into this reactor, without the addition of a glass filter or Received November 23, 1989 Polybrene support. Blots of other sizes can be inserted into this reactor by simply replacing the glass inlet, which carries the bed for the filter, by another of a different bed size. By this means, filter sizes of 4 to 6 x 8 to 12 mm in diameter can be accomodated. Alternatively, we place smaller blot pieces (e. g. 4 5 References x 4 m m ) o n a 6 x 12mmPVDFmembranewithinthereactor. Polybrene treatment is not necessary since the base and the Henschen, A,, Hupe, K.-P., Lottspeich, F. and Voelter, W., 1985, in: acid are delivered (both as liquids) in less than 5 pL quantities High Performance Liquid Chromatography in Biochemistry, VCH Verlagsgesellschaft, Weinheim, Germany. into the reactor bed which allows wetting of the filter(s) but Pearson, I. D., Anal. Biochem. 1986, f52, 189-198. not their complete soaking. This wet-phase-technique 1781 Nice, E. C., Lloyd, C. J. and Burgess, A. W., J . Chromatogr. 1984, enables high coupling and cleavage yields but hinders the pro296, 153-170. tein from being washed out during or after the reactions. Grego, B., Van Driel, I. R., Stearye, P. A., Coding, J. W., Nice, E. C. Therefore, proteins or blots of proteins can be sequenced and Simpson, R. I., Eur. J. Biochem. 1985,148,485-491. directly in this sequencer. Blots derived from smaller peptides Nice, E. C., Grego, B. and Simpson, R. J., Biochem. Int. 1985, 11, are covalently attached to 1,4-phenylene diisocyanate-PVDF 187-195. (MilliGen, Burlington, MA) in the reactor prior to sequence Cooperman, B. S., Weizmann, C. J. and Buck, M. A., Methods Enanalysis (B. Wittmann-Liebold, unpublished results). zymol. 1986,164,523-541.
4.3 Internal amino acid sequence analysis Many proteins are not susceptible to Edman degradation and this is usually attributed to in vivo and/or in vitro amino-terminal blockage of the polypeptide. In these cases, the polypeptide must be fragmented either enzymatically or chemically. The technique developed by Cleveland 1801for in situ digestion of already stained proteins on one-dimensional SDS-PAGE or two-dimensional polyacrylamide gels seems to have some drawbacks due to blockage of the terminal amino groups during electrophoresis or at the fragmentation step (T. Choli, unpublished results). Furthermore, low se-
Kamp, R. M. and Wittmann-Liebold, B., Melhods Enzymol. 1986, 164,542-511. Hunkapiller, M. W., Lujan, E., Ostrander, F. and Hood, L. E., Methods Enzymol. 1983,91, 221-236. Wu, R. S., Stedman,J. D., West, M. H. P., Pantazis, P. and Bonner, W. M., Anal. Biochem. 1982, 124,264-211. Mende, L. M., Waterburg, J. H., Mueller, R. D. and Matthews, H. R., Biochemistry 1983,22, 38-5 1. Waterborg, J. H. and Matthews, H. R., FEES Letf. 1983, 162, 416-4 19. Konigsberg, W. H. and Henderson, L., Melhods Enzymol. 1983,91, 254-259. Baldwin, G . S., Chandler, R., Seet, K. L., Weinstock, I., Grego, B., Rubira, M., Moritz, R. L. and Simpson, R. J., Protein Seq. Data Anal. 1987, I , 7-12.
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[ 141 Ratajczak, T., Camber, M., Moir, R.,Hahuel, R., Grego, B., Rubira,
M. R. and Simpson, R. J., Biochem. Biophys. Res. Commun. 1987, 143,218-224.
! 151 Ratajczak, T., Brockway, M. J., Hahuel, R., Moritz, R. L. and Simpson,R. J.,Biochern. Biophys.Res. Commun. 1988,151,1156-1 163. I161 Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J. and Van Montagu, M., Eur.J. Biochem. 1985, 152,9-19. 1171 Aebersold, R., Teplow, D., Hood, L. E. and Kent, S. B. H.,J. B i d . Chem. 1986,261,4229-4238. I181 Matsudaira, P.,J. B i d . Chem. 1987,262, LOO35-10038. I191 Walsh, M., McDougall J. and Wittmann-Liebold B., Biochemistry 1988,27,6860-6687. 1201 Eckerskorn, C., Mewes, W., Goretzki, H. and Lottspeich, F., Eur. J. Biochem. 1988,176,509-5 19. 1211 Burnette, W. N., Anal. Biochern. 1981,122!, 195-203. 1221 Towbin, H., Staehelin, T. and Gordon, J., Pmc. Natl. Acad. Sci. USA 1979, 76,4350-4354. 1231 Gershoni, J. M. and Palade, G. E., Anal. Biochem. 1982, 124, 396-402. 1241 Laemmli, U. K., Nature 1970,277,680-6131. 1251 Howe, J. G. and Hershey, I. W. B., J. Biol. Chem. 1981, 256, 12836-12839. 1261 Kaltschmidt, E. and Wittmann, H. G., Anal. Biochem. 1970, 36, 401-4 12. 1271 Geyl, D., Bock, A. and Isono, K., Mol. Gen. Genet. 1981, 181, 309-3 12. [281 O’Farrell, P. H., J. B i d . Chem. 1975,250,4007-4021. 1291 Klose, J., Humangenetik 1975,26,231-243. 1301 Visentin, L. P., Chow, C., Matheson, A. T., Yaguchi, M. and Rollin, F., Biochem. J. 1972,130,103-1 10. [3 11 Brockmoller, H. J. and Kamp, R. M., in: Wittmann-Liebold, B., Erd-
mann, V. A. and Salnikow, J. (Eds.), Advanced Methods in Protein Microsequence Analysis, Springer Verlag, Berlin 1986, pp. 34-44. I321 Mets, L. I. and Bogorad, L., AnaL’Biochenz. 1974,57,200-210. [331 Choli, T., Kapp, U. and Wittmann-Liebold, B.,J. Chromatogr. 1989, 476,59-72. 1341 Moos, M., Nguyen, N. Y. and Liu, T-Y., J. Biol. Chem. 1988,263, 6005-6008. 1351 Jovin, T. M., Biochemistry 1973,12, 871-879. [361 Jovin, T. M., Biochemistry 1973,12,879-1390. I371 Jovin, T. M., Biochemistry 1973,12,890-898. t381 Jovin, T. M., Ann. N . Y. Acad. Sci. 1973,209,417-496. [391 Geisthardt,D. and Kruppa, J.,Anal. Biochem. 1987,160,184-191. [401 Morsch, K., Monatsh. Chem. 1988,63,220-235. 1411 Erickson, J. G.,J. Amer. Chem. SOC.1952., 74, 6281-6282. [42J Ogata, Y., Okano, M., Futuya, Y. and Tabuishi, I.,J. Am. Chem. SOC. 1956,78,5426-5428. [431 Friedman, M. and Wall, J. S., J. Amer. Chem. SOC. 1964, 86, 3735-3741. 1966,31,2888-2894. 1441 Friedman,M.andWall,J.S.,J.Org.Chem. [451 Cavins,J. F. andFriedman,M.,Biochemistry 1967,6,3766-3770. [461 Brown, I. R., Fed. Proc. Amer. SOC.Exp. Biol. 1975,34,591-594. 1471 Neurath, H., Cooper, G. R. and Erickson, J. O., J. B i d . Chem. 1942, 142,249. 1481 Klotz, M. I.,Triwush, H. and Walker,F. M..,J. Am. Chem. SOC.1948, 70, 2935. 1491 Kay, M. C. and Edsall, J., Arch. Biochem. Biophys. 1956,65, 354. [501 Klotz,M.I.andStryker,H.V.,J.Am. Chem.Soc. 1960,82,5169. Che,m.Soc. 1963,85,3526. [511 Rupley,J.A.andPraissman,M.,J.Am.
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1521 Gerding,J.J.T.,Koppers, A.,Hagel,P. andBloemendal,H.,Biochim. Biophys. Acta 1971,243,374-379. [531 Hagel, P., Gerding, J. J. T., Fliegen, W. and Bloemendal, H., Biochim. Biophys. Acta 1971,243,366-314. 1541 Shevack, A,, Gewitz, H. S., Henemann, B., Yonath, A. and Witt mann, H. G., FEBS Lett. 1985,184,68-71. [55] Stark, G. R., Biochemistry 1965,4, 1030-1036. [561 Thompson, E. 0. P. and O’Donnell, J., Aust. J. B i d . Sci. 1966,19, 1139-1 15 1. [571 Hardy, S. J. S., Kurland, C. G., Voynow, P. and Mora, G., Biochemistry 1969,8, 2897-2905. [581 Bittner, M., Kuferer, P. and Mouris, C. F.,Anal. Biochem. 1980,102, 459-47 1. 1591 Stellwag, E. J. and Dahlberg, A. E., Nucleic Acids Res. 1980, 8 , 299-3 17. [601 Kyhse-Andersen, J., J . Biochem. Biophys. Methods 1984, 10, 203-209. I611 Pitt-Rivers, R. and Impiombato, F. S. A,, Biochem. J. 1968, 109, 825-839. 1621 Walker, M. J., (Ed.), Methods in Molecular Biology, Humana Press, Clifton, NJ 1984, pp. 165-178. 1631 Reynolds, J. A. and Tanford, C., Proc. Nut. Acad. Sci.USA 1970,66, 1002-1003. 1641 Nelson, C. A.,J. Biol. Chem. 1971,246, 3895-3901. [651 Kapp, 0. H. and Vinogradov, S. N., Anal. Biochem. 1978, 91, 230-235. I661 Poulson, K., Fraser, K. J. and Haber, E., Proc. Natl. Acad. Sci. USA 1972,69,2495-2499. 1671 Hunkapiller, M. W. and Lujan, E., in: Shively, J. E. (Ed.) Methods of Protein Microcharacterization, Humana Press, Clifton, 1986, pp. 89- 101. [681 Wittmann-Liebold, B., Hirano, H. and Kimura, M., in: Wittmann-
Liebold, B., Salnikow, J. and Erdmann, V. (Eds.),AdvancedMethods in Protein Microsequence Analysis, Springer Verlag, Berlin 1986, pp. 77-90. 1691 Eckerskorn, C., Jungblut, P., Mewes, W.,Klose,J. andLottspeich, F., Electrophoresis 1988,9, 830-838. I701 Salinovich, 0. and Montelaro, R. C., Anal. Biochem. 1986, 156, 341-347. 1711 Pluskal, M. F., Przekop, M. B., Kavonian, M. R., Vecoli, C. and Hicks, D. A., BioTechniques 1986,4,272-282. 1721 Yuen, S. W., Chui, A. H., Wilson, K. J. and Yuan, P. M., in: User Bulletin No. 36, Applied Biosystems, Foster City, C A 1988. [731 Bauw, G., Van den Bulke, M., Van Damme, J., Puype, M., Van Montague, M. and Vandekerckhove, J., J . Prof. Chem. 1988, 713, 194. 1988,168,48-53. 1741 Szewczyk,B.andSummers,D.,Anal.Biochem. 1751 Tarr, G. E., Beecher, J. F., Bell, M. and McKearn, D. S., Anal. Biochem. 1978,84,622-627. [761 Klapper, D. C., Wilde, C. E. and Capra, J. D.,Anal. Biochem. 1978, 85, 126-131. [771 Xu, Q:Y. and Shively, E., Anal. Biochem. 1988,170, 19-30. I781 Fischer, S., Reimann, F. and Wittmann, B., in: Wittmann-Liebold, B. (Ed.), Methods in Protein Sequence Analysis, Springer Verlag, Berlin 1989, pp. 98-107. 1791 Wittmann-Liebold, B., J . Prof. Chem. 1988, 7, 304-306. [SO1 Cleveland, D. W., Methods Enzymol. 1983,96,22-229. 1811 Aebersold, R. H., Leavitt, J., Saavedra, A., Hood, L. and Kent, S. B. H., Proc. Natl. Acad. Sci. USA 1987,84,6970-6974.
Electrophoresis 1990,11, 562-568
T. Choli and B. Wittmann-Liebold
Theodora Choli Brigitte Wittmann-Liebold
Protein blotting followed by microsequencing The use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order toprevent N-terminal blockage ofthe proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment for in situ amino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer).
1 Introduction Blotting procedures in combination with microsequencing will continue to be important for the exploration of protein structure. Increased sensitivity in protein analysis has been achieved by systematically improving both the protein purification and sequencing techniques. Although high performance liquid chromatography (HPLC) is widely used for protein isolation [ 1-71 and permits sequence analysis of picomole quantities [SI, sometimes an efficient HPLC resolution for fully purified polypeptide chains cannot be achieved [6,71. Another alternative preparation technique, the electroelution of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or urea gels [8-15], cannot be recommended for polypeptides for subsequent Edman degradation or amino acid analysis, due to coelution of contaminants present in the electrophoresis system. An improvement is the electroblotting of proteins separated by PAGE [8, 17-20]. However, the membranes used for Western blotting 121-231 (commonly referred to as “protein blotting”) e. g., cellulose or nylon derivatives, are chemically unstable and therefore cannot be applied for Edman degradation and amino acid analysis in situ on the membrane. C)n the other hand, derivatized glass filters [16, 17, 201 or Polybrene-coated glass supports [ 161, and synthetic fibers, such as polyvinylidene difluoride (PVDF) [ 18,191 allow in situ NH,-terminal sequencing of proteins, amino acid analysis and determinationof internal sequences after protein blotting. The protein amounts needed for these techniques depend on (i) the type of membrane used, (ii) the hydrophilicity/hydrophobicity of the examined protein, and (iii) the applied sequencing strategy.
2 Blotting of proteins for sequencing 2.1 Prevention of N-terminal blockage PAGE, in combination with blotting to sequencer-stable supports has proven to be a powerful tool for the detection, separation and analysis of proteins. Proteins separated on the basis of their molecular masses [21,24,251 or by two-dimensional electrophoresis [26-321, based om charge differences followed by molecular mass separation in the second dimension, can be further characterized by blotting onto sequencerstable supports such as modified glass fibers (GF) or PVDF Correspondence: Dr. Brigitte Wittmann-Liebold, Max-Planck Institut fur Molekulare Genetik, Abteilung Wittmann, D-1000 Berlin 33 (Dahlem), Germany Abbreviations: PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid (3VCH Verlagsgesellschaft mbH D-6940 Weinheim, 1990
Immobilon (Millipore, Bedford, MA). The elucidation of the primary structure using the Edman chemistry needs a free amino-terminal group. Consequently, the first major consideration for proteins when subjected to electrophoresis, blotting and sequencing by the Edman method is the prevention of any N-terminal modification of the polypeptide chain. A considerable number of precautions should be routinely followed in the preparation of samples to be sequenced by this technique. 2-Mercaptoethanol or dithioerythritol are added to all buffers, used for the isolation and purification steps,in order to avoid formation of aggregates through disulfide bridge formation and the attack of the amino group by radicals, aldehydes or oxygen. T o protect the eluted proteins from possible oxidation, these mercaptans are also used during electrolution. Gel preruns, and the addition of thioglycolic acid (0.1 mM) to the running buffer [ 19, 33, 341 during preelectrophoresis have been reported to increase the initial yields and to prevent tryptophan and methionine from degradation [341.
2.2 Blotting from SDS-PAGE In 1973 Jovin [35-381 published a theory ofmultiphasic zone electrophoresis, making it possible to optimize the choice of buffer system in electrophoresis. According to Geisthard and Kruppa [ 391, amino-containing compounds in the electrophoresis buffer and some of the additives proposed in [381, e. g . thioglycolic acid, react at alkaline pH with residual acrylamide monomers, still present in the polyacrylamide gels after polymerization. Nucleophilic addition of ammonia, primary and secondary amines [40-421, and amino acids 143-441 to a, P-unsaturated carbonylic compounds is a wellknown chemical reaction. Accordingly, the residual acrylamide monomers react with the a-NH,-terminal and the &-NH,-groupsof lysines in proteins and peptides with resultant changes in electrophoretic mobility [391. Alkylation of proteins by acrylamide has also been described [45,461. Strategies to eliminate the accumulation of byproducts in the gel would be beneficial. A preelectrophoresis step and addition of thioglycolic acid to the running buffer would have three effects: (i) removal of charged impurities, (ii) reduction of peroxides and residual radicals, and (iii) scavenging of uncharged reactive species such as acrylamide monomer, free or incorporated acrolein, or other reactive carbonyl compounds. Unfortunately, a preelectrophoresis step in a discontinuous system may result in band diffusion and an impaired separation. Moos and co-workers [34l have suggested a modified, continuous Laemmli system, allowing a separation at pH 7.8 and consequently eliminating the acrylamide monomer reaction with the free a-amino groups of the proteins. This buffer system is recommended for blotting of protein mixtures with closely adjacent bands. A preelectrophoresis step of the nor01 73-0835/90/0707-0562 $3.50+.25/0
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Electroohoresis 1990,Il. 562-568
Figure I. Separation of human heart muscle proteins by one-dimensional (5 %- I 5 %)gradient SDS-PAGE.(A) Protein staining after preelectrophoresis and electrophoresis. (B) Stained protein blot on a PVDF membrane after preelectrophoresis and electrophoresis. ( C )Staining after electrophoresis without preelectrophoresis). Lane (1) marker proteins: (A) 200 kDa; (B) 97.4 kDa;(C) 68 kDa;(D)43 kDa;(E) 29 kDa;(F) 18.4 kDa; (G) 14.3 kDa; lanes (2)-(4) different amounts of heart muscle proteins: 20 pg total protein (left lane and 40 pg (right lane) each. Proteins were electrotransferred onto a PVDF membrane for 1 h using the semidry method with 1.8 mA/cmZ at 4 O C . Staining was done as described in 1331.
mal discontinuous Laemmli system [ 19,331 does not lead to drawbacks if the protein bands in the gel are well resolved. Thus, for optimal performance the SDS gels were prepared a day ahead to reach a high polymerization rate and, depending on the gel size (1 3 x 20 cm or 7 x 8 cm), they were preelectrophoresed at 4 "C overnight or for 2 h, respectively, in a buffer supplemented with 0.01 mM thioglycolic acid. SDS-PAGE of marker and heart muscle proteins, with or without a prerun, did not result in diffusion (Fig. 1) which, however, was observed for other proteins following preelectrophoresis (Fig. 2).
2.3 Blotting from urea gels The interaction of urea with proteins and polypeptides has been studiedextensively [47-5 11. Cyanate andcarbonateions may accumulate in aqueous urea solutions at neutral and alkaline conditions and are most pronounced at a pH > 4-5 [52,53]. The rate of reaction of cyanate with amino groups of proteins appears to involve the unprotonated amine and the unionized cyanic acid: R-NH,
Figure 2. SDS-PAGE (15 % discontinuous system) according to Laemmli 1241 (A) Without and (B) with preelectrophoresis overnight at 4 O C . Left lanes: 50 pg total protein of the ribosome from the archaebacterium Sulfolobus acidocaldurius:right lanes: 10 pg of ribosomal proteins ~ 1 210/ and their fragment (P. Henning, personal communication).
+ NH=C=O + H2O -+ R-NH-CO-NH2 + HZO
This is consistent with the difference in the reaction rates shown by a-amino ( ~ K A 8) = and E-amino groups ( ~ K A 10.7) = of proteins. At pH 7 a-amino groups react 100 times faster than the E-amino groups [551. Carbamylation is not aproblem at p H < 4 and pH > 10I26,561. For this reason ribosomal proteins of E . coli were separated in 6 M urea on CM-cellulose columns under acidic conditions which did not block the N termini of the proteins. The cyanate concentration must be kept low, otherwise the protein will be carbamylated. At low temperatures, the formation of the cyanate ions in urea solutions is extremely slow [52].The acidic ribosomal proteins of Hafobacterium marismortui (Hma-rib proteins) f2, which are only soluble in high molar urea sohtions (6-8 M), ~were maintained in a sequenceable form after solubilization at -20 "C 1191. In addition, the urea used for two-dimensional 5413
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T. Choli and B. Wittmann-Liebold
choice of support and transfer conditions is critical in protein blotting, which is anticipated to provide a true replica of the original gel pattern. The degree to which this is achieved depends mainly on the choice of transfer buffer, the equilibration conditions prior to the transfer and blotting equipment. The electrophoretic transfer of proteins from polyacrylamide gels to the sequencer-stable membranes is performed by two blotting techniques (i) the conventional tank system [22, 58, 591 and (ii) the semi-dry system 1601. For an efficient protein transfer, the entrapment of air bubbles must be avoided in both systems. This can be achieved by pressing the membrane sheet onto the gel surface with a glass rod or spatula. The semi-dry system affords the following advantages over the conventional buffer tank system: (i) The transfer time is drastically reduced and, consequently, the polypeptides are less exposed to oxidation processes. (ii) The filter papers wetted with the buffer are the only buffer reservoirs in the apparatus. (iii) The use of a discontinuous buffer system results in low Joule heat generation 1601. Hence the consumption of buffersin the semidry apparatus is strongly reduced in comparison to the tank version, typically to one tenth of the amount needed in thelatter.
2.5 Preequilibration
Figure 3. Blot onto two consecutive PVDF mernbranes of ribosomal pro teins from the 50s subunit of Bacillus stearothermophilus. Two-dimensional electrophoresis according to 1271; blotting conditions as in 1331. (A) First PVDF membrane; (B) second PVDF membrane.
electrophoresis was of high purity (ultrapure, BRL, Gaithersburg, MD) in order to avoid carbamylation effects. For other ribosomal proteins such as from Bacillus stearothermophilus or from E. coli (Fig. 3) the gel system employed was as describedin 126,271.Theproteinswereextractedfrom70S, 50S, or 30s subunits [571, except that all dialysis buffers contained 10 mM 2-mercaptoethanol. The final dialysis was against 2 % acetic acid with 10 mM 2-mercaptoethanol, which kept the ribosomal proteins from both organisms in a soluble state before lyophilization. The gels were predectrophoresed in the presence of thioglycolic acid for about 3h at 4 "C. After the proteins were electrophoretically transferred onto PVDF membranes [33],they were stained with Amido Black and the excised bands were directly sequenced using the pulse-liquid gas-phase sequencer (Applied Biosysterns, Foster City, C A).
2.4 Electrophoretic transfer The advantage of protein blotting onto membranes is that the transferred proteins are available for further characterization. The proteins are bound to the surface of the sequencer-stable membranes by hydrophobic or electrostatic interactions. The
Independent of the blotting system the gels have to be preequilibrated with the transfer buffer in order to prevent a possible change of buffer conductivity and gel swelling during the transfer, which is performed by applying a constant current. Since proteins are usually eluted from SDS-containing gels, the effect of SDS during transfer and protein immobilization has to be considered. SDS forms a complex with proteins yielding highly negatively charged molecules [6 1I. By using a single transfer buffer for all types of proteins, it is unlikely that all of them will quantitatively be transferred from SDS-PAGE gels, mainly because of the preferential loss of SDS from the gel. As long as SDS remains in the gel, the proteins are in a soluble form. However, owing to its small size and negative charge, SDS migrates out of the gel faster than most proteins, which are then trapped within the gel. Inclusion of SDS in the equilibration buffer considerably improves the transfer efficiency [621.According to I171 the SDS was displaced from the proteins by immersing the gels in 0.5 % v/v Nonidet P-40 for 10 min at room temperature. Most proteins possess two classes of SDS binding sites, i. e. strong binding sites, saturated at 0.4 g SDS/g protein and weaker binding sites saturated at 1.4 g SDS/g protein. A complete removal of SDS from its complexes with proteins is not easy to achieve 163-451. The preequilibration time should not be longer than 10- 15 min in order to avoid band diffusion or leaching of low molecular weight proteins from the gel.
2.6 Transfer buffers Different transfer buffers have been proposed. According to [ 171 the transfer from SDS gels onto chemically activated glass fiber sheets was efficient when 1 % acetic acid [661 and 0.5 % Nonidet P-40 were used. The transfer buffers employed for blotting onto PVDF membranes consist of 25 mM TrisHCI, pH 8.4, 0.02 % SDS and 0.5 % dithiothreitol 119, 331. SDS was added to the transfer buffer in order to improve the transfer of high molecular weight proteins which might otherwise be trapped within the gel. Binding ofthe transferred proteins to the PVDF membrane was not affected by the presence of the detergent (T. Choli, unpublished results) but
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Protein blotting followed by microsequencing
SDS was found to interfere with protein binding to the Polybrene-coated glass fibers and to derivatized nitrocellulose membranes [ 16,621. However, also in the case of the PVDF membranes, the detergent (SDS) concentration in the transfer buffer should be low; otherwise, artifacts will appear during the first few cycles of automated Edman degradation, interfering with the assignment of amino acid residues 1671. In addition, SDS may decrease the pH of the coupling reaction with partial overlaps on degradation as well as slower drying times of the sample after the coupling and cleavage stages 1681.
565
0.1 % Amido Black, in 50 % methanol for 5 min, and destainedin 30 % methanolfor 5-10minatroomtemperature.Staining of PVDF membranes with Coomassie Brilliant Blue or Amido Black leads to lower initial yields in the sequence compared with those of the unstained pieces (see Table 1) [331. After washing and detection, the protein bands are excised and either sequenced immediately or stored at -20 “C under nitrogen in sealed Eppendorf tubes.
3 Sequencing supports The use of methanol, commonly 10-20 %,in the transfer buffers seems to be another critical factor [ 16, 18, 20, 691. According to I 161, swelling of the polyacrylamide gel is avoided and precipitation of the SDS-Polybrene salt on the glass fiber is probably reduced. On the other hand, methanol contains aldehydes which block proteins by formation of Schiffs bases. We did not include methanol in our transfer buffer 19, 331 because no improvement in the transfer efficiency or proteinmembrane binding was noticed with our proteins. The discontinuous buffer used in our experiments has the following composition: the cathode buffer consists of0.04 ~6-amino-n-hexanoic acid, 0.025 M Tris-HC1,20 %v/v methanol, p H 9.4, and that ofthe anode buffer of0.3 MTris-HCl, 20 %v/vmethanol, pH 9.4, and that ofthe anode buffer of0.3 MTris-HCl, 20 % v/ v methanol, pH 10.4, in order to neutralize protons produced at the anode during electrophoresis.
3.1 Glass fiber activation/derivatization
Activated or derivatized glass fibers were the first membranes successfully employed for microsequencing of blotted proteins [ 16, 17, 201. They are activated by immersing in 100 % trifluoracetic acid (TFA) for 1-4 h, drying, and washing in methanol [17, 191. An additional activation step can be performed by immersing the sheets in potassium hydroxide saturated ethanol for 12 h, then in 3 N hydrochloric acid for 12h, or 0.1 %hydrogen fluoride in water for 15 rnin L 191. The activation is followed by derivatization using 3-amino-propyltriethoxysilane or 1,4-phenylene diisothiocyanate [ 171. Polybrene-treated glass fiber membranes are prepared by allowing a solution of 3 mg/mL of Polybrene in water to drip from a Pasteur pipette over the glass fiber sheet. The impregnated sheets are dried in air and are stored at -20 “C. Before use, excess Polybrene is washed out ( 5 rnin with 1 0 0 m L 2.7 Detection of transferred proteins bidistilled water) and the wet sheets are mounted for blotting. After transfer, the membranes are usually washed briefly by Recently, another type of derivatization of glass fiber sheets rinsing in bidistilled water 116, 19, 331,3 x for 10-15 min, to has been reported to show an excellent protein-binding caremove gel contaminants. Proteins blotted onto PVDF mem- pacity [201. According to these researchers the activation branes can be detected without staining as grayish areas on a step was performed by refluxing the sheets in T F A containwhitemembranebackgroundupondrying [16,19).Iflessthan ing 0.2 % poly(methyl-3,3,3-trifluoropropylsiloxane)for 10h 50 pmol of protein per cm2are transferred, they are not visible with gentle stirring. After reaction, the wet sheets were placed [161 and the proteins must be stained with Coomassie Bril- in a desiccator and TFA was removed under vacuum. The liant Blue, Amido Black, Ponceau S [701 or visualized with sheets were then rinsed with acetone toremove excess reagent. fluorescent dyes [ 161. Fluorescamine, reacting with primary Curing of the siloxane linkage was achieved by drying the amino groups to yield fluorescent derivatives I7 11, irreversibly sheets for 60 rnin at 150°C. These sheets are available as blocks a-NH, groups. According to [ 161 this undesirable “Glassy bond” filters (Biometra, Gottingen, FRG). reaction can be avoided by using dilute solutions of 1 mg/200 mL or less. Staining with Ponceau S [701 has the same sen3.2 PVDF membranes sitivity as Coomassie Brilliant Blue R-250 (250 to 500 ng protein), and can be completely removed from the protein by rinsP V D F membranes I181 are mechanically stable, solid-phase ing the stained membrane with water for additional 10-15 supports that bind proteins by hydrophobic interaction. They min. However, its influence on the initial yields of degradation have been used in immunoblotting applications as a substitute has not yet been studied. Most commonly the membranes are for nitrocellulose membranes [721. PVDF membranes are instained with either 0.1 % Coomassie Brilliant Blue R-250 in ert to most solvents (acetonitrile, TFA, ethyl acetate, trimeth50 % methanol, for 5-10 rnin at room temperature, or with ylamine and hexane, but not dimethylformamide), and can be used in automated sequencing. Due to its high hydrophobiciTable 1. Effect of the position of the protein blots in the cartridge block in initial sequencing yield@ ty this membrane should be immersed in 100 % methanol for about 20s [ 181 and equilibrated with the transfer buffer used Detection Arrangement Yield (Yo) for the electrotransfer before placement onto the gel. Coomassie Brilliant Bluebj Amido Blackh, Unstained Unstained
Protein facec) Protein face Protein face Random
24.1 Yo 15.0 % 40.0 % 21.8 %
a) P-Lactoglobulin samples (500 Pmol) were applied onto the gel and blotted after electrophoresis. Initial yields were determined from the NH,terminal residues. The values shown are the percentages ofthe amounts of the protein loaded onto the gel. The yields are average values from three experiments. From 1331 b) Weakly stained for 5 min c) The “protein face” arrangement is depicted in Fig. 4
3.3 Protein binding capacity of various activated and derivatized membranes The above-mentioned membranes have been tested for their binding capacity for standard proteins, i. e. Bst ribosomal proteins (mostly very basic), and Hma (generally acidic) as described in [ 191. The binding capacity has been determined as the 18 amount of protein bound to 1 cm disks of the membranes. The P V D F membranes were found to pos-
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Table 2 Protein binding capacity of sequencer-stable membranes Support
Capacitya)
Activated glass fibers TFA Hydrogen fluoride Hydrochloric acid Potasssium hydroxyde Derivatized supports (glass fibers) Polybrene Aminopropyl Quaternary ammonium Diazotized Diisothiocyanate Polybase-coated glass fibers Polyvinylidene difluoride
5-8 5-8 5-8 5-8 25-35 4-6 4-6 4-6 20-30 30-40
a) Values are given in kg protein/l cm disk of support. The values for activated supports are for proteins blotted from acetic acid-urea gels; the values for the other supports are for proteins blotted from SDS-PAGE gels. From [ 191
sess a higher binding capacity than the derivatized glass membranes, similar to that of Polybrene-treated glass fibers (Table 2).
3.4 Extraction of proteins blotted onto sequencer-stable supports Conditions for extracting proteins blotted onto PVDF or Polybrene-coated glass fiber disks have been reported by several workers [73,741. The efficiency of elution depends on several factors, e. g., hydrophobicity, size of molecule and type of membrane used. It should be noted that once the sample is exposed to sequencing conditions, i. e. after 3 cycles of Edman degradation, the extraction is virtually impossible [721. Proteins blotted onto Polybrene-coated glass fiber sheets can be eluted with 80 % HCOOH [73].. For the extraction of proteins blotted onto PVDF membranes 70 % isopropanol in 5 % TFA was the best volatile solvent combination for adequate extraction (721 with about 60 ‘%proteinrecovery, as assessed by sequencing and determination of initial yields. The proteins were excised and suspended in 0.3 mL of solvent, vortexed and incubated at room temperature for 60 min. The membrane was then removed for sequence analysis, and the solvent was evaporated in a Speed Vac centrifuge and dis-
I+
solved in 30 FL 0.1 % TFA for sequence analysis. Buffer solutions containing SDS and Triton X-100 have also been proposed 1741.Thus, proteins were completely eluted from PVDF membranes at room temperature by short incubation in 50 mM Tris-HC1, p H 9.0, containing 2 % SDS and 1 % Triton X- 100 and the efficiency of elution was practically independent of the molecular weight of the proteins. However, SDS or Triton should be removed from the extracted proteins prior to microsequencing, which is accompanied by further sample loss. Therefore buffers containing detergents or other nonvolatile components should be avoided if the sample is to be used for microsequencing or amino acid analysis.
4 Microsequence analysis 4.1 Microsequence analysis of proteins immobilized on PVDF membranesin the Applied Biosystems sequencer The arrangement in the reactor of pieces of PVDF-blotted protein is an important factor in sequence analysis (Fig. 4) t 19, 331. The membrane surface onto which the protein is blotted (showing the highest intensity) is placed in the upper cartridge in upside-down position, followed by the second PVDF membrane. Both PVDF membranes are kept in position by covering them with a TFA-treated glass fiber, pretreated with 2 mg Polybrene [75,761 andprecycledin anormal sequencing program prior to blotting. The upper cartridge is then turned over and screwed onto the lower cartridge part so that the proteins are on top of the filters. At the R, delivery and wash stages, the blotted proteins are eluted from the PVDF membrane, transferred to and trapped on the pretreated glass filter. With this technique the blotted proteins are degraded mainly in the glass filter employing a normal degradation program. Incomplete coupling due to insufficient penetration of the aqueous base into the PVDF membrane is thereby prevented. According to [771, the glass fiber filter should be removed from the cartridge, otherwise substances may be adsorbed to it instead of to the hydrophobic membrane. Table 1 shows the yield differences between blotted, unstained P-lactoglobulin samples, arranged in the cartridge either with the proteins on top (as described above) or randomly arranged. The initial yields of 40 % in the first case, and 2 1.8 %
a
/
-
1 Membrane
/
Figure 4 . (A) Electrotransfer from an SDSgel onto PVDF membranes. The arrow indicates the blotting direction and (a) shows the membrane surface on which most of the protein was detected. (B) Arrangement of the PVDF pieces in the cartridge of the sequencer. Number 1 and 2 are the first and second PVDF blot, respectively; GF glass fiber disk, pretreated with TFA and precycled with Polybrene.
Electrophoresis 1990,11, 562-568
in the second, demonstrate that the correct sequenceofPVDF membranes is important. Repetitive yields from proteins adsorbed on PVDF membranes were 1-2 % lower compared to that obtained for the same proteins adsorbed to Polybrenetreated fiber filters [ 181. In this case the PVDF membrane was centered onto the Teflon seal without Polybrene-treated glass fibers and placed directly in the cartridge block of the sequencer. The lower initial yields could result from a combination of incomplete cleavage, low coupling, or washing out of peptides. PVDF membranes have a 0.45 pm pore size and are thinner than a glass fiber disk, thus higher liquid volumes are delivered due to the lower back pressure of this membrane. Therefore, too much acid is delivered in the normal degradation program, causing protein loss. The solvent wash steps in the sequencing programs are excessively long for sequencing proteins absorbed onto these membranes. To circumvent these problems and to use the normal sequencing program we prefer to cut the PVDF blot into pieces (a maximum of 3-5 pieces of 4 mm’) which then are placed on top of a precycled Polybrene-treated glass fiber filter. Another efficient approach uses pretreatment of the PVDF membrane with Polybrene, which gave higher initial yields in comparison to a similar electrotransfer on derivatized glass fiber supports 1721. 4.2 Microsequence analysis of proteins immobilized on PVDF membranes in the Knauer sequencer
Protein blotting followed by microsequencing
567
quencing yields are caused by incomplete cleavage in the gel and the formation of several peptides derived from the same protein region. Newer approaches to obtain internal sequence information are to fragment the blotted proteins in situ by proceduresgivenin [33,73,8 11. Inall thesecases,independent of the membrane type used, the membrane pieces (after the transfer) have to be pretreated with polyvinylpyrrolidone (PVP-40) in order to prevent adsorption of the enzyme to the blot-support during digestion. After pretreatment, excess PVP-40 should be removed by extensive washing with water because this chemical strongly absorbs in the UV region. PVDF membranes for blotting were treated with 0.2 % PVP40 in 100 % methanol, followed by incubation in 0.5 % PVP40 in 100 mM acetic acid. Digestion proceeded for 20 h [33], 24 h [731 or overnight (811 at a 1 :20 protein-to-enzyme ratio. For digestion a 100 mM N-methylmorpholine acetate buffer, pH 8.0, is preferable to a bicarbonate buffer or 100 mM TrisHCl, pH 8.5. Carbonate buffers cannot be recommended because they block N-terminal amino groups by Schiff-base formation which becomes a serious disadvantage for blotting of picomole protein quantities. The use of organic solvents 133, 73, 811 in the digestion solution is not always necessary and depends on the protein character and the applied membrane. In case of PVDF blots the presence of 20 % methanol or acetonitrile in the digestion buffer is recommended but is not necessary if an N-methylmorpholine buffer is used [331. For chemical cleavages, e. g. by CNBr treatment of small proteins, the application of PVP-40 can be omitted, while for the fragmentation of bigger molecules its addition is necessary [331.
The Berlin sequencer (Dr. H. Knauer GmbH, Berlin, FRG) has a flow-through reactor [ 781 which can easily accomodate PVDF or glass filter supports. Usually, the oval filters of 6 x 12 mm in diameter are placed into the glass inlet of the reactor followed by protein loading in 5- 10 1 L of 50 % TFA in water. Careful reading of the English version of the manuscript by Blotted protein filters of the same size can be inserted directly Dr. A d a m Inglis is greatly acknowledged. into this reactor, without the addition of a glass filter or Received November 23, 1989 Polybrene support. Blots of other sizes can be inserted into this reactor by simply replacing the glass inlet, which carries the bed for the filter, by another of a different bed size. By this means, filter sizes of 4 to 6 x 8 to 12 mm in diameter can be accomodated. Alternatively, we place smaller blot pieces (e. g. 4 5 References x 4 m m ) o n a 6 x 12mmPVDFmembranewithinthereactor. Polybrene treatment is not necessary since the base and the Henschen, A,, Hupe, K.-P., Lottspeich, F. and Voelter, W., 1985, in: acid are delivered (both as liquids) in less than 5 pL quantities High Performance Liquid Chromatography in Biochemistry, VCH Verlagsgesellschaft, Weinheim, Germany. into the reactor bed which allows wetting of the filter(s) but Pearson, I. D., Anal. Biochem. 1986, f52, 189-198. not their complete soaking. This wet-phase-technique 1781 Nice, E. C., Lloyd, C. J. and Burgess, A. W., J . Chromatogr. 1984, enables high coupling and cleavage yields but hinders the pro296, 153-170. tein from being washed out during or after the reactions. Grego, B., Van Driel, I. R., Stearye, P. A., Coding, J. W., Nice, E. C. Therefore, proteins or blots of proteins can be sequenced and Simpson, R. I., Eur. J. Biochem. 1985,148,485-491. directly in this sequencer. Blots derived from smaller peptides Nice, E. C., Grego, B. and Simpson, R. J., Biochem. Int. 1985, 11, are covalently attached to 1,4-phenylene diisocyanate-PVDF 187-195. (MilliGen, Burlington, MA) in the reactor prior to sequence Cooperman, B. S., Weizmann, C. J. and Buck, M. A., Methods Enanalysis (B. Wittmann-Liebold, unpublished results). zymol. 1986,164,523-541.
4.3 Internal amino acid sequence analysis Many proteins are not susceptible to Edman degradation and this is usually attributed to in vivo and/or in vitro amino-terminal blockage of the polypeptide. In these cases, the polypeptide must be fragmented either enzymatically or chemically. The technique developed by Cleveland 1801for in situ digestion of already stained proteins on one-dimensional SDS-PAGE or two-dimensional polyacrylamide gels seems to have some drawbacks due to blockage of the terminal amino groups during electrophoresis or at the fragmentation step (T. Choli, unpublished results). Furthermore, low se-
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