ANAI.YTICAL

BIOCHEMISTRY

Visualization

98, 81-84

(1979)

of Proteins Two-Dimensional

after Isoelectric Focusing Gel Electrophoresis

HERBERT

Received

during

JACKLE

January

26. 1979

A modification of P. H. O’Farrell’s (1975,5. Viol. Chrrrr. 259,4007-4021) two-dimensional gel electrophoresis is described. After isoelectric focusing, the cylindrical gels were fixed and stained with Coomassie brilliant blue R before sodium dodecyl sulfate-slab gel electrophoresis in the second dimension. The modification does not alter the protein patterns obtained, but provides sharper spots. In addition, bands are made visible before separation in the second dimetnjion. Moreover, the modification helps to reduce the amount of ampholine in the dye front during electrophoresis.

make sure that the wanted proteins were included, one had to cut out a region larger than the desired part because the exact position varies slightly in different runs. This indirect procedure is now avoided by staining the gels after isoelectric focusing. Following the staining procedure described here, gels can also be stored for several weeks at room temperature without any loss of resolution.

In the two..dimensional gel electrophoresis introduced by O’Farrell (1,2), isoelectric focusing of the proteins in a cylindrical gel (first dimension) is followed by electrophoresis in an SDS-slab’gel (second dimension). Before being loaded onto the slab gel, isoelectric focusing gels are equilibrated in SDS buffer in order to prevent streaking of proteins in the second dimension. The equilibration procedure, however, results in a loss of 5 to 25% of the total proteins and in a defocusing of bands due to diffusion. These problems are overcome by using nonequilibrated gels, but this leads to a streaking of high molecular weight proteins. Up to now no method has been described for visualizing protein bands after isoelectric focusing and before separation in the SDSslab gel. In comparing proteins of a distinct isoelectric plaint but from different focusing gels in one SDS-slab gel. methods for detecting the desired position have always been indirect. i.e., dividing the isoelectric focusing gel into longitudinal parts or calibration with the aid of reference gels. To ’ Abbreviation used: SDS. sodium TEMED. tetra~ethylmethylenediamine.

dodecyl

MATERIALS

AND METHODS

ML~~(JP~N~s.Egg clusters and females, after egg deposition, were taken from a laboratory stock of Smitfiu spy. (Chironomidae, Diptera) as described by Kalthoff and Sander (3). Ultrapure urea was purchased from Schwarz/Mann: ampholine from LKB; acrylamide, N’R;“-methylenebisacrylamide, and SDS from Polyscience; RNase A, Tris. histidine. Coomassie brilliant blue R, pmercaptoethanol. Nonidet P-40, glycine, lysine, TEMED and all salts from Sigma. Srrrr~plr preparcrtion. Eggs and females were sonicated for 30 min in 20 PI 0.001 M Tris-HCI. pH 7.4. containing 1 mg RNase A/ml and 5 mM MgCI?. In order to pellet

sulfate:

81

0003-2697179113008 Copyright All nghtr

I-04$02.00/0

E 1979 by Academic Pres,. Inc. of reproductw ,n any form re\ervcd.

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HERBERTJACKLE

egg debris, yolk particles. and chitin parts of the females, samples were centrifuged for 20 min at 12,000~~at 2°C. The supernatant was diluted with 20 PI 0.05 M lysine, containing 0.5% SDS (w/v), and adjusted to 9.5 M urea by adding solid urea. Finally. the sample was diluted with I vol of lysis buffer (9.5 M urea, 10% P-mercaptoethanol, 4%~Nonidet P-40, 3% ampholine, pH 5-8, and 1% ampholine, pH 3-10). Electsophorrsis techniquc>s. Samples containing 15 to 60 pg protein were loaded onto 0.25 X II-cm isoelectric focusing gels prepared according to O’Farrell (1). The upper reservoir of the gel tank was filled with 0.01 M histidine instead of NaOH. Gels were run for 14 h at 400 V and then for 1 additional h at 800 V with an LKB 2103 power supply. Slab gels contained 0.375 M TrisHCl (pH 8.8), 0.1% SDS, 9.8% acrylamide, 0.2% N’N’-methylenebisacrylamide, 0.15% TEMED. and 0.025% ammonium persulfate. The stacking gel was 2.5 cm long and contained 4% acrylamide, 5% glycerol, 0.15% TEMED, 0.1% SDS (w/v), 0.025%~ ammonium persulfate, and 0.1 M Tris-HCI (pH 6.5). Gels were prepared according to Studier (4) and run at a constant current (30 mA) in buffer made from 133 g glycine. 30 g TrisilO liters distilled water containing 0.1% SDS (w/v). Runs were completed after the dye front (bromphenol blue, 0.005%) had moved 9.5 cm into the separation gel. Fixation undstcrit~ir~~. Isoelectric focusing gels were fixed and stained for 10 min in 0.1% Coomassie brilliant blue R dissolved in an aqueous solution containing 50%, methanol and 10% acetic acid. Slab gels were stained in the same solution for at least 2 h. Destaining for both gels was carried out for 1 h in 50% methanol containing 10% acetic acid followed by 5% methanol in 7% acetic acid, containing 0.005% Coomassie brilliant blue R to prevent total destaining. Equilihtxrtim. Prior to electrophoresis in the second dimension, gels were placed in

5 ml equilibration buffer(0.625 M Tris-HCI, 2.3% SDS (w/v), 5% p-mercaptoethanol. IOP glycerol. pH 6.5) for 30 min three times. RESULTS The major proteins from Smittitr eggs were resolved using the two-dimensional gel electrophoresis of O’Farrell (I). The original technique was modified in order to achieve optimal resolution of the proteins studied. In the sample buffer, SDS was used for better solubilization of the remaining yolk proteins (see arrows in Fig. 1) while lysine prevented artificial charge heterogeneity of proteins by carbamylation, which might be caused by isocyanate formed from the decomposition of urea. The cathodic drift of the pH gradient during isoelectric focusing was minimized by using 0.01 M histidine instead of NaOH as electrolyte solution in the upper reservoir of the gel tank (5). The pattern of proteins obtained with this method after reduction of ampholines in the dye front is shown in Fig. 1 (top). Proteins were fixed and stained after isoelectric focusing, and some of the gels were then stored up to 6 weeks at room temperature. After equilibration. gels were applied to the second dimension (2). When the current was switched on, Coomassie brilliant blue inside the isoelectric focusing gel became stacked and moved with the bromphenol dye front through the stacking gel, and in the interspace between the dye and the SDS front in the separation gel. The Coomassie brilliant blue, released from the formerly stained proteins in the isoelectric focusing gel, was still visible as dark and somewhat broadened “bands” in front of the bromphenol blue front. After staining and destaining overnight, no proteins could be detected in the isoelectric focusing gel, on top of the slab gel, or in front of the bromphenol blue front. However, the pattern of proteins as shown in

VISUALIZATION

OF PROTEINS

Fig. I (top) became visible (Fig. 1, bottom). Almost no alterations could be detected, except that ampholines had disappeared except for a faint line in the bromphenol blue front. In order to compare specific sections from different isoelectric focusing gels. instead of running the total protein patterns on different SDS-slab gels, the bands of interest from the stained isoelectric focusing gels can be cut out and then electrophoresed in the same slab gel. For this purpose, stained isoelectric focusing gels were fixed in 50% methanol in 10% acetic acid. After this treatment, gels became more compact and bands more stacked. The region of interlest could now be cut out using a razor blade without the necessity of external reference points (see introduction). If very thin sections were desired, two razor blades were bolted together using washers or Teflon strips as spacers, and bands were cut out after freezing the gel at -70°C. Slices of about 1 mm could easily be obtained and processed. Each slice was equilibrated individually in 50 ~1 of equilibration buffer per millimeter of slice length for 30 min three times and placed on top of the SDS-slab gel as usual (1). This procedure allows one to compare even single bands from differnt isoelectric focusing gels. In order to demonstrate this, a dominant band from a stained isoelectric focusing gel of egg proteins and its corresponding band from female proteins were separated in one SDS-slab gel (Fig. 1, bottom, inset). The protein patterns obtained with the modification described here were the same as those achieved with the original method of O’Farrell (1.2) and even provide a sharpening of spots (Fig. 1). The position of protein spots was also reproducible and not affected by storage of the isoelectric focusing gels after fixation and staining. This has been observed when one band from 1.5 different isoelectric focusing gets that contained the same amount of protein

AFTER

ISOELECTRIC

83

FOCUSING

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FIG. 1. Protein pattern after two-dimensional gel electrophoresis of the major proteins from eggs of Stnirtirr sprc. The l?.OOOg supernatant was prepared as described under Materials and Methods. and 40 ~1 samples were separated in the isoelectric focusing dimension (IEF) followed by resolution in the SDS dimension (SDS) in a 10% polyacrylamide gel. Gels were stained as described under Materials and Methods. Asterisks indicate the position of the dye front in the SDS-slab gels. (Top) Original method: The amount of ampholines was reduced by soaking the stained gels in 50% alcohol, 7% acetic acid. 0.005% Coomassie brilliant blue for 45 h. (Bottom) Modification: Isoelectric focusing gels were fixed and stained prior to resolution in the SDS dimension. The box indicates a major yolk component region of the gel (arrow). The corresponding band to one major yolk component (arrow) from stained isoelectric focusing gels each containing 60 pg total protein of eggs and females, respectively. was cut out and coelectrophorized in one SDS-slab gel. A, Eggs: B. females.

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and had been stored over different periods up to 6 weeks were compared on one slab gel. DISCUSSION The modification of the improved twodimensional gel electrophoresis technique (l,2) described here makes proteins in isoelectric focusing gels visible prior to electrophoresis in SDS-slab gels. During SDS-slab gel electrophoresis all visible stain disappeared from the proteins, which then behave as unstained proteins during electrophoresis. In addition, staining of ampholine in the dye front is drastically reduced with processing of the stained SDSslab gel (I), due to the disappearance of ampholines from the isoelectric focusing gel during fixation and staining procedure. The reason for their loss is the fact that ampholines behave like very small proteins ( 1) and are not precipitated with acid in the absence of SDS. They diffuse out of the isoelectric focusing gel within a few hours (unpublished observation). In contrast, SDS is always present in unfixed and unstained isoelectric focusing gels during equilibration and/or SDS-slab gel electrophoresis. Therefore, ampholines become acid precipitable, remain in the gel, and are stained with common protein stains. Moreover, regions with a distinct pH value or even narrow bands from different isoelectric focusing gels can now be compared after electrophoresis on one SDS-

JACKLE

slab gel directly. If radioactively labeled proteins in nonstainable amounts are to be compared, proteins with known isoelectric points may serve as internal markers. Finally, this method may simplify preparative purification of proteins. Large amounts of proteins can be focused in the first purification step without overloading the gel. After fixation and staining, narrow bands can be cut out from a number of gels and separated on one second-dimension slab gel. ACKNOWLEDGMENTS I thank Dr. Klaus Kalthoff for stimulation and critical discussion during the course of this work. and f or critically reading the manuscript. I thank Dr. James K. Skipper for reading the manuscript and Mrs. Joan Hunter for linguistic help. This work was supported by Grant No. AI 15046. awarded to Dr. Klaus Kalthoff by the U. S. National Institute of Allergy and Infectious Diseases. I am indebted to the Deutsche Forschungsgemeinschaft for a research fellowship.

REFERENCES 1. O’Farrell, P. H. (1975lJ. Biol. Chew 259, 40074021. 2. O’Farrell, P. H., and O’Farrell. P. 2.. (1977) irr Methods in Cell Biology (Stein, G., Stein, J.. and Kleinsmith. L. J., eds.), Vol. XVI. pp. 407-420, Academic Press, New York. 3. Kalthoff, K.. and Sander. K. (1968) Wilhe/m Rou.r’ Arch. Ent~t~ic~~/un~sn~~~~~h. Or~crnisrn~r~ 161, l29- 146. 4. Studier, F. W. t 1973) J. MI)/. Biol. 79, 237-248. 5. Nguyen, N. Y., and Chrambach. A. (1977) And. Biochrm. 82, 54-62.

Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis.

ANAI.YTICAL BIOCHEMISTRY Visualization 98, 81-84 (1979) of Proteins Two-Dimensional after Isoelectric Focusing Gel Electrophoresis HERBERT Rec...
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