Chapter 24 Western Blotting Using PVDF Membranes and Its Downstream Applications Setsuko Komatsu Abstract Western blotting using polyvinylidene difluoride (PVDF) membranes is one of the most popular techniques for detection and characterization of proteins. If this technique is combined with immunodetection, the behavior of a particular protein can be clarified. On the other hand, if it is combined with Edman sequencing, the primary structure of the protein can be determined. A protein sample is transferred from an SDS-polyacrylamide gel electrophoresis (PAGE) gel onto a PVDF membrane by electroblotting. The membrane carrying the protein is either used for immunodetection or protein sequencing. SDS-PAGE followed by Western blotting combined with immunodetection using antibodies can easily detect protein behavior in crude protein mixtures. Furthermore, two-dimensional PAGE followed by Western blotting and Edman sequencing allows effective sequence determination of crude protein mixtures that may not be easily purified by conventional column chromatography. Key words Western blotting, Edman sequencing, PVDF membrane, Immunodetection, Deblocking, Cleveland method
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Introduction Western blotting is a widely accepted analytical technique used to detect and identify specific proteins in a crude protein extract. Gel electrophoresis is first used to separate structural or denatured proteins. The proteins are transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane, where they are allowed to react with an antibody specific to the target protein, which often involves staining for identification. Although methods without an electrophoresis step have been improved, Western blotting based on an immunoreaction is widely used in the fields of molecular biology, biochemistry, immunogenetics and related disciplines. Edman sequencing of proteins separated on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels became possible with the introduction of protein electroblotting methods that allow efficient transfer of sample from the gel matrix onto
Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_24, © Springer Science+Business Media New York 2015
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supports suitable for gas-phase sequencing or related techniques [1]. Picomole amounts of protein are first separated by 2D-PAGE [2], and then electroblotted from 2D-PAGE gels onto a PVDF membrane. The amino acid sequence of the electroblotted protein is determined by Edman sequencing. Direct N-terminal sequencing is the most sensitive method (1–5 μg of protein), but when gaps or ambiguous assignments are seen, verification of the sequence by other means often demands much more material. Proteins are often posttranslationally modified, and N-terminal blockage is one of the more common posttranslational modifications. Proteins can become N-terminally blocked not only in vivo but also in vitro. However, it is possible to prevent in vitro blocking, which is caused during protein extraction, 2D-PAGE, and blotting. The use of very pure reagents during these procedures, the addition of thioglycolic acid as a free radical scavenger to the extraction buffer, electrophoresis, and electroblotting buffers, and preelectrophoresis to remove free radicals from the gel may all be effective in preventing in vitro blocking [3]. However, if proteins are blocked in vivo, a chemical or enzymatic deblocking procedure or peptide mapping procedure is required to determine the N-terminal or internal sequence.
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Materials 1. Sodium dodecyl sulfate (SDS) sample buffer: 60 mM Tris– HCl (pH 6.8), 2 % SDS, 10 % glycerol, and 5 % β-mercaptoethanol [4]. 2. Acrylamide for the separating gel (acrylamide/bisacrylamide = 30:0.135): 30.00 g acrylamide, 0.135 g bisacrylamide. Make volume to 100 mL with Milli-Q water, and keep in the dark (brown bottle). 3. Separating gel buffer (pH 8.8): 12.11 g Tris–HCl to a 1 M final concentration, 0.27 g SDS to a 0.27 % final concentration. Dissolve in 80 mL Milli-Q water, adjust pH to 8.8, and make the volume to 100 mL. 4. Acrylamide for stacking gel (acrylamide/bisacrylamide = 29.2:0.8): 29.2 g acrylamide, 0.8 g bis-acrylamide. Make volume to 100 mL with Milli-Q water, and keep in the dark (brown bottle). 5. Stacking gel buffer (pH 6.8): 3.03 g Tris–HCl to a 0.25 M final concentration, 0.20 g SDS to a 0.2 % final concentration. Dissolve in 80 mL Milli-Q water, adjust the pH to 6.8, and make the volume to 100 mL. 6. SDS-PAGE running buffer: 9 g Tris–HCl, 43.2 g glycine, and 3 g SDS. Dissolve in 3 L Milli-Q water. 7. Bromophenol blue (BPB) solution: Dissolve 0.1 g BPB and 10 g glycerol in 100 mL Milli-Q water.
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8. Blotting buffer A: 36.33 g Tris–HCl to a 0.3 M final concentration, 200 mL methanol to a 20 % final concentration, and 0.20 g SDS to a 0.02 % final concentration. Make the volume to 1 L with Milli-Q water, and keep at 4 °C. 9. Blotting buffer B: 3.03 g Tris–HCl to a 25 mM final concentration, 200 mL methanol to a 20 % final concentration, and 0.20 g SDS to a 0.02 % final concentration. Make the volume to 1 L with Milli-Q water, and keep at 4 °C. 10. Blotting buffer C: 3.03 g Tris–HCl to a 25 mM final concentration, 5.20 g ε-aminocaproic acid to a 40 mM final concentration, 200 mL methanol to a 20 % final concentration, and 0.20 g SDS to a 0.02 % final concentration. Make the volume to 1 L with Milli-Q water, and keep at 4 °C. 11. Separating gel solution (amounts are for one gel [18 %]): 10 mL acrylamide for separating gel, 6.3 mL separating gel buffer (pH 8.8), 120 μL 10 % ammonium persulfate (APS), and 20 μL TEMED. 12. Stacking gel solution (amounts are for one gel [5 %]): 1 mL acrylamide for stacking gel, 3 mL stacking gel buffer (pH 6.8), 2 mL Milli-Q water, 30 μL 10 % APS, and 20 μL TEMED. 13. Hydration buffer: Make this buffer with 8 M urea, 2 % w/v CHAPS, 50 mM DTT, 0.2 % ampholyte (pH 3.5–10) and a trace of 0.001 % BPB.
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Methods
3.1 Two-Dimensional Polyacrylamide Gel Electrophoresis
For 2D-PAGE, there are two options for the separation in the first dimension: immobilized pH gradient (IPG) strips or isoelectric focusing tubes. The first option is described here.
3.1.1 Separation in Immobilized pH Gradient Strips as the First Dimension
1. Use a nonlinear IPG strip (pI 3.5–10.0, 18 cm) for separation in the first dimension. It offers high resolution, great reproducibility and allows high protein loads. 2. Hydrate the strips overnight in the preswelling cassette with 25 mL hydration buffer. 3. When the rehydration cassette is thoroughly emptied and opened, transfer the strips to the strip tray. After placing IPG strips, humid electrode wicks, electrodes, and sample cups in position, cover the strips and cups with low-viscosity paraffin oil. Apply samples slowly at the cathode end of the IPG strips and continue this manner without touching the strip gels. 4 Increase the voltage linearly from 300 to 3,500 V over 3 h, followed by three additional hours at 3,500 V, and then increase it to 5,000 V. Total volt-h will be 8–10,000 V h. 5. After separation using IPG strips, perform SDS-PAGE in the second dimension.
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3.1.2 SDS-PAGE as the Second Dimension
1. Clip together two glass plates (100 × 140 × 1 mm) with a clip, keeping a 1-mm space between the plates. 2. Prepare separating gel solution in a 100-mL beaker. Mix the solutions and fill the plates to about 2 cm from the top. (Caution: Pour the solutions into the plates immediately after adding 10 % APS and TEMED.) 3. Overlay the separating gel solution with 1 mL Milli-Q water. 4. Leave the gel for 40–60 min at room temperature for polymerization. 5. Remove the overlaid water. 6. Prepare the stacking gel solution in a 100-mL beaker. Mix well and pour on the separating gel. 7. Leave the gel for 20 min at room temperature for polymerization. 8. Apply the first-dimension IPG strip directly on the top of the stacking gel. Overlay the first dimension strip with 1 % agarose. 9. Assemble the slab gel for electrophoresis. Add a few drops of BPB in the SDS-PAGE running buffer. 10. Run the sample at 35 mA (constant current) until the tracking dye reaches the bottom of the separating gel. 11. Separate the stacking gel using cutter, and take out the separation gel for the next step.
3.2 Cleveland Peptide Mapping [5]
First separate the samples by 2D-PAGE. Then stain the gels with Coomassie brilliant blue (CBB), and remove the gel pieces (5–20 pieces) containing protein spots and soak them for 1 h in Milli-Q water in a 2 mL microcentrifuge tube. Remove the Milli-Q water and add 750 μL electroelution buffer. Electroelute the protein from the gel pieces using an electrophoretic concentrator run at 2 W constant power for 2 h. After electroelution, dialyze the protein solution against Milli-Q water for 48 h and lyophilize them.
3.2.1 Peptide Mapping Protocol
1. Cut out stained protein spots from 2D gels and soak them in Milli-Q water for 1 h. 2. Fill the 2-mL Eppendorf tube containing the protein spots (5–20 gel pieces) with 750 μL electroelution buffer. Shake for 30 min. 3. Cut seamless cellulose tubing (small size, no. 24, Wako, Osaka, Japan) into pieces 12- to 15-cm long, as space is needed for clipping. Fill a 300 mL beaker with 250 mL Milli-Q water, boil it for 5 min, and keep the tubing membrane in it after boiling. Wet small piece of cellophane film in a small beaker with Milli-Q water.
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b
Large part
Small part
Cellophane film Twisting Gel pieces are deposited.
Tubing membrane
Clipping
Tubing membrane is twisted.
Fig. 1 Electrophoretic concentrator. (a) Close-up of the cup with long seamless cellulose tubing. (b) The cups fixed to the electrophoretic concentrator. (Reproduced from Komatsu [7] with permission from Springer)
4. Close the bottom of the small part of the cup of electrophoretic concentrator with cellophane film, open the bottom of the large part of the cup, connect and twist the tubing membrane, and close the distal end by clipping (Fig. 1a). 5. Fix the cup in the electrophoretic concentrator (Nippon-Eido, Tokyo, Japan) (Fig. 1b). Deposit gel pieces containing proteins on the cellophane film (the small part of the cup), and add 750 μL of electroelution buffer from the Eppendorf tube. Fill the small part of the cup with electroelution buffer, and then fill the large part of the cup with electroelution buffer in such a way that a layer of buffer joins both parts of the cup, allowing movement of protein from the small part of the cup to the tubing membrane. Fill the apparatus with electroelution buffer. The small part of the cup containing the protein spots should be toward the positive side. 6. Run at 2 W constant power for 2 h. 7. Remove the tubing membrane and clip to close the end. Dialyze in a cold room (4 °C). Change the Milli-Q water three times the first day. The next day, change the Milli-Q water two times. 8. Transfer the protein solution to two to six 2 mL microcentrifuge tubes. Freeze-dry overnight. 9. Dissolve the protein in 20 μL SDS sample buffer.
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3.2.2 V8 Protease Digestion
Dissolve the protein in 20 μL SDS sample buffer (pH 6.8) and apply it to a sample well of an SDS-PAGE gel. Overlay the sample solution with 20 μL of a solution containing 10 μL Staphylococcus aureus V8 protease (Pierce, Rockford, IL, USA) (0.1 μg/μL) in Milli-Q water and 10 μL SDS sample buffer (pH 6.8). Electrophorese until the sample and protease are stacked in the stacking gel. Switch off the power for 30 min to allow digestion of the protein, and continue electrophoresis. A detailed procedure follows. 1. Clip together two glass plates (100 × 140 × 1 mm) with a clip, keeping a 1-mm space between the plates. 2. Prepare separating gel solution in a 100-mL beaker. Mix the solutions and fill the plates to about 3 cm from the top. (Caution: Pour the solutions into the plates immediately after adding 10 % APS and TEMED.) 3. Overlay the separating gel solution with 1 mL Milli-Q water. 4. Leave the gel for 40–60 min at room temperature for polymerization. 5. Remove the overlaid water. 6. Prepare the stacking gel solution in a 100-mL beaker. Mix well, pour on the separating gel, and insert comb. 7. Leave the gel for 20 min at room temperature for polymerization. 8. Take out the comb, clips, and silicon tubes as spacer. 9. Clean the wells using Milli-Q water with a syringe. 10. Insert the gel plates into the apparatus. Pour SDS-PAGE running buffer into the apparatus. 11. Dissolve the protein in 20 μL SDS sample buffer (pH 6.8), and apply to a sample well of the SDS-PAGE gel. Overlay the sample with 20 μL of a solution containing 10 μL Staphylococcus aureus V8 protease at 1 μg/μL in Milli-Q water and 10 μL SDS sample buffer (pH 6.8). Add 30 μL BPB solution. 12. Electrophorese until the sample and protease are stacked in the upper gel and then interrupt the run for 30 min to digest the protein. 13. Run the gel at 35 mA until the BPB line reaches about 5 mm from the bottom. 14. Disconnect the electrical leads, and take out the plates. 15. Separate the two plates with a spatula. 16. Separate the stacking gel, and take out the separating gel for the next step.
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Fig. 2 Western blotting. Following separation by 2D-PAGE or by the Cleveland method, the proteins in the gel are electroblotted onto a PVDF membrane using a semidry transfer blotter. Layers of Whatman 3MM filter paper are wetted in A, B and C blotting buffer, and gel and PVDF membrane are wetted in C blotting buffer. (Reproduced from Komatsu [7] with permission from Springer)
3.3
Western Blotting
Following separation by 2D-PAGE or by the Cleveland method, electroblot the proteins onto a PVDF membrane (Fig. 2) using a semidry transfer blotter, and detect the proteins by CBB staining. 1. Cut the PVDF membrane to a size equal to that of the gel. 2. Cut Whatman 3MM filter paper to a size equal to that of the gel. 3. Wash the PVDF membrane in methanol for a few seconds, and transfer the membrane to 100 mL blotting buffer C, and shake for 5 min. 4. Wet two sheets of Whatman 3MM filter paper in blotting buffer A, B or C (blotting paper A, B or C). 5. Place the separating gel in 100 mL blotting buffer C and shake for 5 min. 6. Wet the semidry transfer blotter with Milli-Q water. Place blotting paper A on the blotting plate followed by blotting paper B. Remove air bubbles, if any. Place the PVDF membrane on the plate followed by the gel and blotting paper C. 7. Connect the power supply. Run the blot at 1 mA/cm2 for 90 min. 8. Wash the PVDF membrane in 100 mL Milli-Q water. 9. Stain the PVDF membrane for 2–3 min in CBB. 10. Destain the PVDF membrane in 60 % methanol for 3 min twice. 11. Wash with Milli-Q water and air-dry at room temperature.
3.4 Deblocking of Blotted Proteins [6] (See Note 1) 3.4.1 Acetylserine and Acetylthreonine
For proteins separated by 2D-PAGE that have an acetylserine or acetylthreonine block at their N-termini, first electroblot the gel onto a PVDF membrane. Excise the region of the PVDF membrane carrying the protein spot and treat with trifluoroacetic acid at 60 °C for 30 min. These samples can then be sequenced directly (see Note 2).
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3.4.2 Formyl Group
For proteins separated by 2D-PAGE that are blocked due to N-formylation, first electroblot the gel onto a PVDF membrane. Excise the region of the PVDF membrane carrying the protein spot and treat with 300 μL 0.6 M HCl at 25 °C for 24 h. Wash the membrane with Milli-Q water, dry it, and apply it to the protein sequencer.
3.4.3 Pyroglutamic Acid
The following procedure will remove pyroglutamic acid from the N-termini of proteins. 1. After separation by 2D-PAGE, electroblot proteins with pyroglutamic acid at their N-termini onto a PVDF membrane. 2. Excise the region of the PVDF membrane carrying the protein spot and treat with 200 μL 0.5 % (w/v) polyvinylpyrrolidone40 in 100 mM acetic acid at 37 °C for 30 min (see Note 3). 3. Wash the PVDF membrane at least ten times with 1 mL Milli-Q water. 4. Soak the PVDF membrane in 100 μL 0.1 M phosphate buffer (pH 8) containing 5 mM dithiothreitol and 10 mM EDTA. 5. Add pyroglutamyl peptidase (5 μg), and incubate the reaction solution at 30 °C for 24 h. 6. Wash the PVDF membrane with Milli-Q water, dry it, and apply it to the protein sequencer [6].
3.5 N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search of Amino Acid Sequence
1. Excise the stained protein spots or bands from the PVDF membrane and apply to the upper glass block of the reaction chamber of a gas-phase protein sequencer, such as Procise 494 or cLC (Applied Biosystems, Foster City, CA, USA) or PPSQ (Shimazu, Kyoto, Japan). Perform Edman degradation according to the standard program supplied by Applied Biosystems or Shimazu. Separate the released phenylthiohydantoin amino acids by an online high-performance liquid chromatography system and identify them by retention time. 2. Compare the amino acid sequences obtained with those of known proteins in the Swiss-Prot, PIR, GenPept, and PDB databases with the web-accessible search program FastA.
3.6 Immune Reaction with Antibody 3.6.1 Blocking and Incubating
1. Incubate the membrane with blocking buffer on a shaker for 1–2 h at 37 °C or overnight at 4 °C. 2. Dilute primary antibody with primary antibody dilution buffer and incubate the membrane with the diluted primary antibody on a shaker for 1 h at 37 °C or overnight at 4 °C. 3. Wash the membrane four times with washing buffer on the shaker for 10 min each time. 4. Dilute secondary antibody with blocking buffer and incubate the membrane with the diluted secondary antibody conjugated
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ADH
M
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Fig. 3 Example of immune blotting. Proteins were extracted from soybean, separated by SDS-PAGE, Western blotted, incubated with anti-alcohol dehydrogenase antibody, and detected with the ECL method. CBB staining was used to control for loading
with horseradish peroxidase (HRP) on a shaker for 1 h at 37 °C or overnight at 4 °C. 5. Wash the membrane four times with washing buffer on the shaker for 10 min each time. 3.6.2 Detection of HRP-Conjugated Secondary Antibody
4
1. Detect protein with an ECL kit. In a separate tube, mix black and white ECL solutions in a 1:1 ratio. 2. Aliquot solution onto membranes and wait for 1 min. Drain the ECL, wrap the membrane in plastic and expose it to film. Expose the blots for 10 s, 1 min, 5 min, and more to visualize the chemiluminescence signal that corresponds to the specific antibody-antigen reaction (Fig. 3).
Notes 1. These deblocking techniques may be combined to allow the sequential deblocking and sequencing of unknown proteins that have been immobilized onto PVDF membranes. A protein on the PVDF membrane can be directly used for gas-phase sequencing. If sequencing fails at this step, remove the PVDF membrane from the sequencer, remove the acetyl group, the formyl group, and then the pyroglutamic group. 2. The advantage of this method is that deblocking is easy and rapid, although overall sequencing yields obtained by this procedure are low compared with acylamino acid-releasing enzyme digestion. N-acetylated proteins can be enzymatically deblocked with acylamino acid-releasing enzyme after on-membrane
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digestion with trypsin to generate an N-terminal peptide fragment. This tryptic digestion is required since acylamino acidreleasing enzyme can only remove the acylamino acid from a short peptide [6]. 3. Polyvinylpyrrolidone-40 is used to unbind pyroglutamic acid from the PVDF membrane, while the rest of the protein stays bound to it.
Acknowledgement The author thanks Mr. Yin at the National Institute of Crop Science for providing results of Western blotting. References 1. Eckerskorn C, Mewes W, Goretzki H, Lottspeich F (1988) A new siliconized-gas fiber as support for protein-chemical analysis of electroblotted proteins. Eur J Biochem 176:509–512 2. O’Farrell PF (1975) High resolution twodimensional electrophoresis of proteins. J Biol Chem 250:4007–4021 3. Hirano H, Komatsu S, Kajiwara H, Takagi Y, Tsunasawa S (1993) Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting. Electrophoresis 14:839–846 4. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
5. Cleveland DW, Fischer SG, Krischer MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J Biol Chem 252:1102–1106 6. Hirano H, Komatsu S, Nakamura A et al (1991) Structural homology between semidwarfismrelated proteins and glutelin seed protein in rice (Oryza sativa L.). Theor Appl Genet 83: 153–158 7. Komatsu S (2009) Western blotting/Edman sequencing using PVDF membrane. In: Kurien BT, Scofield RH (eds) Protein blotting and detection, vol 536, Methods in molecular biology. Springer, New York, pp 163–171
1
Introduction Western blotting is a widely accepted analytical technique used to detect and identify specific proteins in a crude protein extract. Gel electrophoresis is first used to separate structural or denatured proteins. The proteins are transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane, where they are allowed to react with an antibody specific to the target protein, which often involves staining for identification. Although methods without an electrophoresis step have been improved, Western blotting based on an immunoreaction is widely used in the fields of molecular biology, biochemistry, immunogenetics and related disciplines. Edman sequencing of proteins separated on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels became possible with the introduction of protein electroblotting methods that allow efficient transfer of sample from the gel matrix onto
Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_24, © Springer Science+Business Media New York 2015
227
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Setsuko Komatsu
supports suitable for gas-phase sequencing or related techniques [1]. Picomole amounts of protein are first separated by 2D-PAGE [2], and then electroblotted from 2D-PAGE gels onto a PVDF membrane. The amino acid sequence of the electroblotted protein is determined by Edman sequencing. Direct N-terminal sequencing is the most sensitive method (1–5 μg of protein), but when gaps or ambiguous assignments are seen, verification of the sequence by other means often demands much more material. Proteins are often posttranslationally modified, and N-terminal blockage is one of the more common posttranslational modifications. Proteins can become N-terminally blocked not only in vivo but also in vitro. However, it is possible to prevent in vitro blocking, which is caused during protein extraction, 2D-PAGE, and blotting. The use of very pure reagents during these procedures, the addition of thioglycolic acid as a free radical scavenger to the extraction buffer, electrophoresis, and electroblotting buffers, and preelectrophoresis to remove free radicals from the gel may all be effective in preventing in vitro blocking [3]. However, if proteins are blocked in vivo, a chemical or enzymatic deblocking procedure or peptide mapping procedure is required to determine the N-terminal or internal sequence.
2
Materials 1. Sodium dodecyl sulfate (SDS) sample buffer: 60 mM Tris– HCl (pH 6.8), 2 % SDS, 10 % glycerol, and 5 % β-mercaptoethanol [4]. 2. Acrylamide for the separating gel (acrylamide/bisacrylamide = 30:0.135): 30.00 g acrylamide, 0.135 g bisacrylamide. Make volume to 100 mL with Milli-Q water, and keep in the dark (brown bottle). 3. Separating gel buffer (pH 8.8): 12.11 g Tris–HCl to a 1 M final concentration, 0.27 g SDS to a 0.27 % final concentration. Dissolve in 80 mL Milli-Q water, adjust pH to 8.8, and make the volume to 100 mL. 4. Acrylamide for stacking gel (acrylamide/bisacrylamide = 29.2:0.8): 29.2 g acrylamide, 0.8 g bis-acrylamide. Make volume to 100 mL with Milli-Q water, and keep in the dark (brown bottle). 5. Stacking gel buffer (pH 6.8): 3.03 g Tris–HCl to a 0.25 M final concentration, 0.20 g SDS to a 0.2 % final concentration. Dissolve in 80 mL Milli-Q water, adjust the pH to 6.8, and make the volume to 100 mL. 6. SDS-PAGE running buffer: 9 g Tris–HCl, 43.2 g glycine, and 3 g SDS. Dissolve in 3 L Milli-Q water. 7. Bromophenol blue (BPB) solution: Dissolve 0.1 g BPB and 10 g glycerol in 100 mL Milli-Q water.
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8. Blotting buffer A: 36.33 g Tris–HCl to a 0.3 M final concentration, 200 mL methanol to a 20 % final concentration, and 0.20 g SDS to a 0.02 % final concentration. Make the volume to 1 L with Milli-Q water, and keep at 4 °C. 9. Blotting buffer B: 3.03 g Tris–HCl to a 25 mM final concentration, 200 mL methanol to a 20 % final concentration, and 0.20 g SDS to a 0.02 % final concentration. Make the volume to 1 L with Milli-Q water, and keep at 4 °C. 10. Blotting buffer C: 3.03 g Tris–HCl to a 25 mM final concentration, 5.20 g ε-aminocaproic acid to a 40 mM final concentration, 200 mL methanol to a 20 % final concentration, and 0.20 g SDS to a 0.02 % final concentration. Make the volume to 1 L with Milli-Q water, and keep at 4 °C. 11. Separating gel solution (amounts are for one gel [18 %]): 10 mL acrylamide for separating gel, 6.3 mL separating gel buffer (pH 8.8), 120 μL 10 % ammonium persulfate (APS), and 20 μL TEMED. 12. Stacking gel solution (amounts are for one gel [5 %]): 1 mL acrylamide for stacking gel, 3 mL stacking gel buffer (pH 6.8), 2 mL Milli-Q water, 30 μL 10 % APS, and 20 μL TEMED. 13. Hydration buffer: Make this buffer with 8 M urea, 2 % w/v CHAPS, 50 mM DTT, 0.2 % ampholyte (pH 3.5–10) and a trace of 0.001 % BPB.
3
Methods
3.1 Two-Dimensional Polyacrylamide Gel Electrophoresis
For 2D-PAGE, there are two options for the separation in the first dimension: immobilized pH gradient (IPG) strips or isoelectric focusing tubes. The first option is described here.
3.1.1 Separation in Immobilized pH Gradient Strips as the First Dimension
1. Use a nonlinear IPG strip (pI 3.5–10.0, 18 cm) for separation in the first dimension. It offers high resolution, great reproducibility and allows high protein loads. 2. Hydrate the strips overnight in the preswelling cassette with 25 mL hydration buffer. 3. When the rehydration cassette is thoroughly emptied and opened, transfer the strips to the strip tray. After placing IPG strips, humid electrode wicks, electrodes, and sample cups in position, cover the strips and cups with low-viscosity paraffin oil. Apply samples slowly at the cathode end of the IPG strips and continue this manner without touching the strip gels. 4 Increase the voltage linearly from 300 to 3,500 V over 3 h, followed by three additional hours at 3,500 V, and then increase it to 5,000 V. Total volt-h will be 8–10,000 V h. 5. After separation using IPG strips, perform SDS-PAGE in the second dimension.
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3.1.2 SDS-PAGE as the Second Dimension
1. Clip together two glass plates (100 × 140 × 1 mm) with a clip, keeping a 1-mm space between the plates. 2. Prepare separating gel solution in a 100-mL beaker. Mix the solutions and fill the plates to about 2 cm from the top. (Caution: Pour the solutions into the plates immediately after adding 10 % APS and TEMED.) 3. Overlay the separating gel solution with 1 mL Milli-Q water. 4. Leave the gel for 40–60 min at room temperature for polymerization. 5. Remove the overlaid water. 6. Prepare the stacking gel solution in a 100-mL beaker. Mix well and pour on the separating gel. 7. Leave the gel for 20 min at room temperature for polymerization. 8. Apply the first-dimension IPG strip directly on the top of the stacking gel. Overlay the first dimension strip with 1 % agarose. 9. Assemble the slab gel for electrophoresis. Add a few drops of BPB in the SDS-PAGE running buffer. 10. Run the sample at 35 mA (constant current) until the tracking dye reaches the bottom of the separating gel. 11. Separate the stacking gel using cutter, and take out the separation gel for the next step.
3.2 Cleveland Peptide Mapping [5]
First separate the samples by 2D-PAGE. Then stain the gels with Coomassie brilliant blue (CBB), and remove the gel pieces (5–20 pieces) containing protein spots and soak them for 1 h in Milli-Q water in a 2 mL microcentrifuge tube. Remove the Milli-Q water and add 750 μL electroelution buffer. Electroelute the protein from the gel pieces using an electrophoretic concentrator run at 2 W constant power for 2 h. After electroelution, dialyze the protein solution against Milli-Q water for 48 h and lyophilize them.
3.2.1 Peptide Mapping Protocol
1. Cut out stained protein spots from 2D gels and soak them in Milli-Q water for 1 h. 2. Fill the 2-mL Eppendorf tube containing the protein spots (5–20 gel pieces) with 750 μL electroelution buffer. Shake for 30 min. 3. Cut seamless cellulose tubing (small size, no. 24, Wako, Osaka, Japan) into pieces 12- to 15-cm long, as space is needed for clipping. Fill a 300 mL beaker with 250 mL Milli-Q water, boil it for 5 min, and keep the tubing membrane in it after boiling. Wet small piece of cellophane film in a small beaker with Milli-Q water.
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b
Large part
Small part
Cellophane film Twisting Gel pieces are deposited.
Tubing membrane
Clipping
Tubing membrane is twisted.
Fig. 1 Electrophoretic concentrator. (a) Close-up of the cup with long seamless cellulose tubing. (b) The cups fixed to the electrophoretic concentrator. (Reproduced from Komatsu [7] with permission from Springer)
4. Close the bottom of the small part of the cup of electrophoretic concentrator with cellophane film, open the bottom of the large part of the cup, connect and twist the tubing membrane, and close the distal end by clipping (Fig. 1a). 5. Fix the cup in the electrophoretic concentrator (Nippon-Eido, Tokyo, Japan) (Fig. 1b). Deposit gel pieces containing proteins on the cellophane film (the small part of the cup), and add 750 μL of electroelution buffer from the Eppendorf tube. Fill the small part of the cup with electroelution buffer, and then fill the large part of the cup with electroelution buffer in such a way that a layer of buffer joins both parts of the cup, allowing movement of protein from the small part of the cup to the tubing membrane. Fill the apparatus with electroelution buffer. The small part of the cup containing the protein spots should be toward the positive side. 6. Run at 2 W constant power for 2 h. 7. Remove the tubing membrane and clip to close the end. Dialyze in a cold room (4 °C). Change the Milli-Q water three times the first day. The next day, change the Milli-Q water two times. 8. Transfer the protein solution to two to six 2 mL microcentrifuge tubes. Freeze-dry overnight. 9. Dissolve the protein in 20 μL SDS sample buffer.
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3.2.2 V8 Protease Digestion
Dissolve the protein in 20 μL SDS sample buffer (pH 6.8) and apply it to a sample well of an SDS-PAGE gel. Overlay the sample solution with 20 μL of a solution containing 10 μL Staphylococcus aureus V8 protease (Pierce, Rockford, IL, USA) (0.1 μg/μL) in Milli-Q water and 10 μL SDS sample buffer (pH 6.8). Electrophorese until the sample and protease are stacked in the stacking gel. Switch off the power for 30 min to allow digestion of the protein, and continue electrophoresis. A detailed procedure follows. 1. Clip together two glass plates (100 × 140 × 1 mm) with a clip, keeping a 1-mm space between the plates. 2. Prepare separating gel solution in a 100-mL beaker. Mix the solutions and fill the plates to about 3 cm from the top. (Caution: Pour the solutions into the plates immediately after adding 10 % APS and TEMED.) 3. Overlay the separating gel solution with 1 mL Milli-Q water. 4. Leave the gel for 40–60 min at room temperature for polymerization. 5. Remove the overlaid water. 6. Prepare the stacking gel solution in a 100-mL beaker. Mix well, pour on the separating gel, and insert comb. 7. Leave the gel for 20 min at room temperature for polymerization. 8. Take out the comb, clips, and silicon tubes as spacer. 9. Clean the wells using Milli-Q water with a syringe. 10. Insert the gel plates into the apparatus. Pour SDS-PAGE running buffer into the apparatus. 11. Dissolve the protein in 20 μL SDS sample buffer (pH 6.8), and apply to a sample well of the SDS-PAGE gel. Overlay the sample with 20 μL of a solution containing 10 μL Staphylococcus aureus V8 protease at 1 μg/μL in Milli-Q water and 10 μL SDS sample buffer (pH 6.8). Add 30 μL BPB solution. 12. Electrophorese until the sample and protease are stacked in the upper gel and then interrupt the run for 30 min to digest the protein. 13. Run the gel at 35 mA until the BPB line reaches about 5 mm from the bottom. 14. Disconnect the electrical leads, and take out the plates. 15. Separate the two plates with a spatula. 16. Separate the stacking gel, and take out the separating gel for the next step.
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Fig. 2 Western blotting. Following separation by 2D-PAGE or by the Cleveland method, the proteins in the gel are electroblotted onto a PVDF membrane using a semidry transfer blotter. Layers of Whatman 3MM filter paper are wetted in A, B and C blotting buffer, and gel and PVDF membrane are wetted in C blotting buffer. (Reproduced from Komatsu [7] with permission from Springer)
3.3
Western Blotting
Following separation by 2D-PAGE or by the Cleveland method, electroblot the proteins onto a PVDF membrane (Fig. 2) using a semidry transfer blotter, and detect the proteins by CBB staining. 1. Cut the PVDF membrane to a size equal to that of the gel. 2. Cut Whatman 3MM filter paper to a size equal to that of the gel. 3. Wash the PVDF membrane in methanol for a few seconds, and transfer the membrane to 100 mL blotting buffer C, and shake for 5 min. 4. Wet two sheets of Whatman 3MM filter paper in blotting buffer A, B or C (blotting paper A, B or C). 5. Place the separating gel in 100 mL blotting buffer C and shake for 5 min. 6. Wet the semidry transfer blotter with Milli-Q water. Place blotting paper A on the blotting plate followed by blotting paper B. Remove air bubbles, if any. Place the PVDF membrane on the plate followed by the gel and blotting paper C. 7. Connect the power supply. Run the blot at 1 mA/cm2 for 90 min. 8. Wash the PVDF membrane in 100 mL Milli-Q water. 9. Stain the PVDF membrane for 2–3 min in CBB. 10. Destain the PVDF membrane in 60 % methanol for 3 min twice. 11. Wash with Milli-Q water and air-dry at room temperature.
3.4 Deblocking of Blotted Proteins [6] (See Note 1) 3.4.1 Acetylserine and Acetylthreonine
For proteins separated by 2D-PAGE that have an acetylserine or acetylthreonine block at their N-termini, first electroblot the gel onto a PVDF membrane. Excise the region of the PVDF membrane carrying the protein spot and treat with trifluoroacetic acid at 60 °C for 30 min. These samples can then be sequenced directly (see Note 2).
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3.4.2 Formyl Group
For proteins separated by 2D-PAGE that are blocked due to N-formylation, first electroblot the gel onto a PVDF membrane. Excise the region of the PVDF membrane carrying the protein spot and treat with 300 μL 0.6 M HCl at 25 °C for 24 h. Wash the membrane with Milli-Q water, dry it, and apply it to the protein sequencer.
3.4.3 Pyroglutamic Acid
The following procedure will remove pyroglutamic acid from the N-termini of proteins. 1. After separation by 2D-PAGE, electroblot proteins with pyroglutamic acid at their N-termini onto a PVDF membrane. 2. Excise the region of the PVDF membrane carrying the protein spot and treat with 200 μL 0.5 % (w/v) polyvinylpyrrolidone40 in 100 mM acetic acid at 37 °C for 30 min (see Note 3). 3. Wash the PVDF membrane at least ten times with 1 mL Milli-Q water. 4. Soak the PVDF membrane in 100 μL 0.1 M phosphate buffer (pH 8) containing 5 mM dithiothreitol and 10 mM EDTA. 5. Add pyroglutamyl peptidase (5 μg), and incubate the reaction solution at 30 °C for 24 h. 6. Wash the PVDF membrane with Milli-Q water, dry it, and apply it to the protein sequencer [6].
3.5 N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search of Amino Acid Sequence
1. Excise the stained protein spots or bands from the PVDF membrane and apply to the upper glass block of the reaction chamber of a gas-phase protein sequencer, such as Procise 494 or cLC (Applied Biosystems, Foster City, CA, USA) or PPSQ (Shimazu, Kyoto, Japan). Perform Edman degradation according to the standard program supplied by Applied Biosystems or Shimazu. Separate the released phenylthiohydantoin amino acids by an online high-performance liquid chromatography system and identify them by retention time. 2. Compare the amino acid sequences obtained with those of known proteins in the Swiss-Prot, PIR, GenPept, and PDB databases with the web-accessible search program FastA.
3.6 Immune Reaction with Antibody 3.6.1 Blocking and Incubating
1. Incubate the membrane with blocking buffer on a shaker for 1–2 h at 37 °C or overnight at 4 °C. 2. Dilute primary antibody with primary antibody dilution buffer and incubate the membrane with the diluted primary antibody on a shaker for 1 h at 37 °C or overnight at 4 °C. 3. Wash the membrane four times with washing buffer on the shaker for 10 min each time. 4. Dilute secondary antibody with blocking buffer and incubate the membrane with the diluted secondary antibody conjugated
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ADH
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Fig. 3 Example of immune blotting. Proteins were extracted from soybean, separated by SDS-PAGE, Western blotted, incubated with anti-alcohol dehydrogenase antibody, and detected with the ECL method. CBB staining was used to control for loading
with horseradish peroxidase (HRP) on a shaker for 1 h at 37 °C or overnight at 4 °C. 5. Wash the membrane four times with washing buffer on the shaker for 10 min each time. 3.6.2 Detection of HRP-Conjugated Secondary Antibody
4
1. Detect protein with an ECL kit. In a separate tube, mix black and white ECL solutions in a 1:1 ratio. 2. Aliquot solution onto membranes and wait for 1 min. Drain the ECL, wrap the membrane in plastic and expose it to film. Expose the blots for 10 s, 1 min, 5 min, and more to visualize the chemiluminescence signal that corresponds to the specific antibody-antigen reaction (Fig. 3).
Notes 1. These deblocking techniques may be combined to allow the sequential deblocking and sequencing of unknown proteins that have been immobilized onto PVDF membranes. A protein on the PVDF membrane can be directly used for gas-phase sequencing. If sequencing fails at this step, remove the PVDF membrane from the sequencer, remove the acetyl group, the formyl group, and then the pyroglutamic group. 2. The advantage of this method is that deblocking is easy and rapid, although overall sequencing yields obtained by this procedure are low compared with acylamino acid-releasing enzyme digestion. N-acetylated proteins can be enzymatically deblocked with acylamino acid-releasing enzyme after on-membrane
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digestion with trypsin to generate an N-terminal peptide fragment. This tryptic digestion is required since acylamino acidreleasing enzyme can only remove the acylamino acid from a short peptide [6]. 3. Polyvinylpyrrolidone-40 is used to unbind pyroglutamic acid from the PVDF membrane, while the rest of the protein stays bound to it.
Acknowledgement The author thanks Mr. Yin at the National Institute of Crop Science for providing results of Western blotting. References 1. Eckerskorn C, Mewes W, Goretzki H, Lottspeich F (1988) A new siliconized-gas fiber as support for protein-chemical analysis of electroblotted proteins. Eur J Biochem 176:509–512 2. O’Farrell PF (1975) High resolution twodimensional electrophoresis of proteins. J Biol Chem 250:4007–4021 3. Hirano H, Komatsu S, Kajiwara H, Takagi Y, Tsunasawa S (1993) Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting. Electrophoresis 14:839–846 4. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
5. Cleveland DW, Fischer SG, Krischer MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J Biol Chem 252:1102–1106 6. Hirano H, Komatsu S, Nakamura A et al (1991) Structural homology between semidwarfismrelated proteins and glutelin seed protein in rice (Oryza sativa L.). Theor Appl Genet 83: 153–158 7. Komatsu S (2009) Western blotting/Edman sequencing using PVDF membrane. In: Kurien BT, Scofield RH (eds) Protein blotting and detection, vol 536, Methods in molecular biology. Springer, New York, pp 163–171
