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Drying and storage of polyacrylamide slab gels: A simple procedure
ANALYTICAL
BIOCHEMISTRY
163,42-44
Drying and Storage
(1987)
of Polyacrylamide BABRU BAHAN
Slab Gels: A Simple Procedure SAMAL
Amgen, 1900 Oak Terrace Lane, Thousand Oaks, California 91320 Received October 2, 1986 A simple procedure for drying and storing of polyacrylamide slab gels is described. A polyacrylamide slab gel is fixed in acetic acid plus glycerol and then sandwiched between a gel bond plastic sheet and a dialysis membrane in the presence of a minute amount of gelatin and dried on the benchtop at room temperature. The fixed gel can be stored indefinitely. 0 1987 Academic Fks,
Inc.
KEY WORDS: electrophoresis; protein; storage; gelatin; dialysis membrane; gel-fix sheets.
procedure (1). For silver staining, the gel was processed as follows: 15 min twice in 40% methanol + 10% acetic acid, 10 min twice in 10% ethanol + 5% acetic acid; 15 min in a freshly made DTT (Sigma) solution (5 mg/ liter); 5 min in distilled water; 20 min in freshly prepared silver nitrate (Kodak) solution (1.72 g/liter); 5 min in distilled water. Staining was developed in a solution of 0.3 M sodium carbonate (Sigma) + 0.0185% formaldehyde (Mallinckrodt). The gel was then fixed and temporarily stored in 10% glycerol + 10% acetic acid prior to drying. Five grams of gelatin (Sigma) was suspended in a final volume of 100 ml in water and then autoclaved for 30 min at 121 “C and 20 lb pressure to make the solution homogeneous. The gelatin solution was stored at room temperature, but was routinely warmed to 50-55°C before use. A plastic sheet (gel-fix for PAGE, 265 X 193 X 0.18 mm, No. 42983, Serva Feinbiochemica, Heidelberg) was placed on a flat surface, and 3-4 ml of warm gelatin solution was pipetted onto the center of the sheet. The gel to be stored was removed from the acetic MATERIALS AND METHODS acid-glycerol solution, excess solution was A standard SDS’-polyacrylamide slab gel allowed to drip off, and then the gel was (12.5%) was run according to the published placed on the gel-fix sheet. A thin film of gelatin was left between the interface of the ’ Abbreviations used: SDS, sodium dodecyl sulfate; gel-fix and the gel (I 10 X 150 X 1 mm) by DTT, dithiothreitol; PAGE, polyacrylamide gel electrothis process. Care was taken not to trap any phoresis.
Electrophoresis of proteins on a slab gel containing different percentages of polyacrylamide is widely used for resolving proteins of different molecular weights (l-3). After the gel is stained with Coomassie blue or silver nitrate, either a black-and-white or a color photograph is taken for recordkeeping purposes, and then the gel is dried or stored either at 4°C or at room temperature in acetic acid. It has been difficult and time consuming to dry and store a large number of gels for future observation. A commercial slab gel dryer is generally used for this purpose; however, its use necessitates a vacuum pump, dry ice, and a heating unit. Usually only one gel can be dried at a time. If there is loss of vacuum during the drying process, the gel is often shattered. In this report, a simple procedure in which a number of gels can be dried simultaneously and then stored for future reference is described. There is no need for a gel dryer, vacuum pump, or dry ice. This method has been used for the past several years with excellent results.
OOO3-2697187 $3.00 CopyrightQ 1987byAcademic AU rights of reproduction
FVas, Inc. in any form rewwd.
42
DRYING
AND STORAGE
OF POLYACRYLAMIDE
air bubbles in the interface. On the top of the gel about 3 ml of gelatin was added and left to spread evenly. One piece of dialysis memNo. brane (165 X 185 mm, Bio-Rad, 165-0922), which was soaked in 10% acetic acid-lo% glycerol buffer, was spread on the top of the gel, thus trapping a minimum amount of gelatin. The membrane was always larger than the gel, so that there was direct adhesion of the membrane to the gelfix sheet on all four sides of the gel. Adhesion was accomplished by the presence of the gelatin solution, which upon drying, formed a thin film and acted as a glue. Any air bubbles present were removed by carefully lifting the dialysis membrane from one side and slowly spreading it on the gel and the gel-fix. Excess
SLAB GELS
43
gelatin from the gel-fix was removed with Kimwipe to prevent one gel-fix from sticking to another when they were stored. The amount of gelatin trapped in the gel-fix/gel and gel/dialysis membrane and gel-fix/dialysis membrane interfaces was about 3-4 ml. Four 1OO-ml water-filled bottles or equivalent weights (about 200-300 g) were placed on four corners of the gel-fix sheet to prevent it from curling during drying. The bottles were placed away from the dialysis membrane gel-fix interface. Because the amount of gelatin trapped was minimal over a large area, there was no need for a fan or heat. After 48 h, the pliable gel was ready to be stored conveniently in a three-ring binder after holes were punched in the gel-fix sheet,
,
II
OA CA
STI
LA
FIG. 1. A 12.5% polyacrylamide-SDS slab gel was run, stained, dried, and stored as described under Materials and Methods. The relative positions of the dialysis membrane (E) covering the gel and the bottles (A, B, C, D) on gel-fix sheet (F) are indicated. Molecular weight standards are PB, phosphorylase b; BSA, bovine serum albumin; OV, ovalbumin; CA, carbonic anhydrase, STI, soybean trypsin inhibitor; LA, ol-lactalbumin. The samples were from hepatitis purification steps.
1
44
BABRU BAHAN
away from the area covered by the dialysis membrane. RESULTS AND DISCUSSION
Figure 1 is representative of the polyacrylamide slab gel that had been silver stained, dried, and stored in the manner described in this paper. This and other gels have been kept intact for more than 2 years. The number of gels that have cracked during storage is minimal. For example, out of 253 gels processed during the last few years, only 13 were cracked. Polyacrylamide gels of different percentages (7.5 to 15%) used for gel electrophoresis or isoelectric focusing and stained with either silver nitrate or Coomassie blue have been stored using this procedure. Gel bond sheet (FMC) instead of gel-fix (Serva) could also be used for storing dried gels. Although isoelectric focusing gels could be dried otherwise, this method provides a means for long-term storage in which both sides of the gel are protected. Stained agarose gels could also be treated in a similar manner for long-term storage. Agarose gels may be dried by other means but the method described in this paper ensures their durability upon storage.
SAMAL
The work toward this procedure was inspired by publications of 0. Popescu (4). Juang et al. (5) also described another method for gel drying after our process was initially developed. The major advantage of the current procedure is the use of gel-fix sheets and a dialysis membrane to seal the gel in gelatin, so that dried gels can be stored side-by-side in a binder without sticking to each other. If necessary, the stored gels can easily be scanned with a densitometer at any time. ACKNOWLEDGMENTS I thank Mark Zukowski, Barbara Karan, and Evryll Swanson for help during the preparation of this manuscript and Joan Bennett for typing the manuscript.
REFERENCES 1. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 2. O’Farrell, P. Z., Goodman, H. M., and O’Farrell, P. H. (1977) Cell 12, 1133-I 142. 3. Hoffman, W. L., and Dowben, R. M. (1978) Anal. Biochem. 89,540-549. 4. Popescu, 0. (1983) Electrophoresis 4,432-433. 5. Juang, R.-H., Chang, Y.-D., Sung, H.-Y., and Su, J.-C. (1984) Anal. Biochem. 141,348-350.
BIOCHEMISTRY
163,42-44
Drying and Storage
(1987)
of Polyacrylamide BABRU BAHAN
Slab Gels: A Simple Procedure SAMAL
Amgen, 1900 Oak Terrace Lane, Thousand Oaks, California 91320 Received October 2, 1986 A simple procedure for drying and storing of polyacrylamide slab gels is described. A polyacrylamide slab gel is fixed in acetic acid plus glycerol and then sandwiched between a gel bond plastic sheet and a dialysis membrane in the presence of a minute amount of gelatin and dried on the benchtop at room temperature. The fixed gel can be stored indefinitely. 0 1987 Academic Fks,
Inc.
KEY WORDS: electrophoresis; protein; storage; gelatin; dialysis membrane; gel-fix sheets.
procedure (1). For silver staining, the gel was processed as follows: 15 min twice in 40% methanol + 10% acetic acid, 10 min twice in 10% ethanol + 5% acetic acid; 15 min in a freshly made DTT (Sigma) solution (5 mg/ liter); 5 min in distilled water; 20 min in freshly prepared silver nitrate (Kodak) solution (1.72 g/liter); 5 min in distilled water. Staining was developed in a solution of 0.3 M sodium carbonate (Sigma) + 0.0185% formaldehyde (Mallinckrodt). The gel was then fixed and temporarily stored in 10% glycerol + 10% acetic acid prior to drying. Five grams of gelatin (Sigma) was suspended in a final volume of 100 ml in water and then autoclaved for 30 min at 121 “C and 20 lb pressure to make the solution homogeneous. The gelatin solution was stored at room temperature, but was routinely warmed to 50-55°C before use. A plastic sheet (gel-fix for PAGE, 265 X 193 X 0.18 mm, No. 42983, Serva Feinbiochemica, Heidelberg) was placed on a flat surface, and 3-4 ml of warm gelatin solution was pipetted onto the center of the sheet. The gel to be stored was removed from the acetic MATERIALS AND METHODS acid-glycerol solution, excess solution was A standard SDS’-polyacrylamide slab gel allowed to drip off, and then the gel was (12.5%) was run according to the published placed on the gel-fix sheet. A thin film of gelatin was left between the interface of the ’ Abbreviations used: SDS, sodium dodecyl sulfate; gel-fix and the gel (I 10 X 150 X 1 mm) by DTT, dithiothreitol; PAGE, polyacrylamide gel electrothis process. Care was taken not to trap any phoresis.
Electrophoresis of proteins on a slab gel containing different percentages of polyacrylamide is widely used for resolving proteins of different molecular weights (l-3). After the gel is stained with Coomassie blue or silver nitrate, either a black-and-white or a color photograph is taken for recordkeeping purposes, and then the gel is dried or stored either at 4°C or at room temperature in acetic acid. It has been difficult and time consuming to dry and store a large number of gels for future observation. A commercial slab gel dryer is generally used for this purpose; however, its use necessitates a vacuum pump, dry ice, and a heating unit. Usually only one gel can be dried at a time. If there is loss of vacuum during the drying process, the gel is often shattered. In this report, a simple procedure in which a number of gels can be dried simultaneously and then stored for future reference is described. There is no need for a gel dryer, vacuum pump, or dry ice. This method has been used for the past several years with excellent results.
OOO3-2697187 $3.00 CopyrightQ 1987byAcademic AU rights of reproduction
FVas, Inc. in any form rewwd.
42
DRYING
AND STORAGE
OF POLYACRYLAMIDE
air bubbles in the interface. On the top of the gel about 3 ml of gelatin was added and left to spread evenly. One piece of dialysis memNo. brane (165 X 185 mm, Bio-Rad, 165-0922), which was soaked in 10% acetic acid-lo% glycerol buffer, was spread on the top of the gel, thus trapping a minimum amount of gelatin. The membrane was always larger than the gel, so that there was direct adhesion of the membrane to the gelfix sheet on all four sides of the gel. Adhesion was accomplished by the presence of the gelatin solution, which upon drying, formed a thin film and acted as a glue. Any air bubbles present were removed by carefully lifting the dialysis membrane from one side and slowly spreading it on the gel and the gel-fix. Excess
SLAB GELS
43
gelatin from the gel-fix was removed with Kimwipe to prevent one gel-fix from sticking to another when they were stored. The amount of gelatin trapped in the gel-fix/gel and gel/dialysis membrane and gel-fix/dialysis membrane interfaces was about 3-4 ml. Four 1OO-ml water-filled bottles or equivalent weights (about 200-300 g) were placed on four corners of the gel-fix sheet to prevent it from curling during drying. The bottles were placed away from the dialysis membrane gel-fix interface. Because the amount of gelatin trapped was minimal over a large area, there was no need for a fan or heat. After 48 h, the pliable gel was ready to be stored conveniently in a three-ring binder after holes were punched in the gel-fix sheet,
,
II
OA CA
STI
LA
FIG. 1. A 12.5% polyacrylamide-SDS slab gel was run, stained, dried, and stored as described under Materials and Methods. The relative positions of the dialysis membrane (E) covering the gel and the bottles (A, B, C, D) on gel-fix sheet (F) are indicated. Molecular weight standards are PB, phosphorylase b; BSA, bovine serum albumin; OV, ovalbumin; CA, carbonic anhydrase, STI, soybean trypsin inhibitor; LA, ol-lactalbumin. The samples were from hepatitis purification steps.
1
44
BABRU BAHAN
away from the area covered by the dialysis membrane. RESULTS AND DISCUSSION
Figure 1 is representative of the polyacrylamide slab gel that had been silver stained, dried, and stored in the manner described in this paper. This and other gels have been kept intact for more than 2 years. The number of gels that have cracked during storage is minimal. For example, out of 253 gels processed during the last few years, only 13 were cracked. Polyacrylamide gels of different percentages (7.5 to 15%) used for gel electrophoresis or isoelectric focusing and stained with either silver nitrate or Coomassie blue have been stored using this procedure. Gel bond sheet (FMC) instead of gel-fix (Serva) could also be used for storing dried gels. Although isoelectric focusing gels could be dried otherwise, this method provides a means for long-term storage in which both sides of the gel are protected. Stained agarose gels could also be treated in a similar manner for long-term storage. Agarose gels may be dried by other means but the method described in this paper ensures their durability upon storage.
SAMAL
The work toward this procedure was inspired by publications of 0. Popescu (4). Juang et al. (5) also described another method for gel drying after our process was initially developed. The major advantage of the current procedure is the use of gel-fix sheets and a dialysis membrane to seal the gel in gelatin, so that dried gels can be stored side-by-side in a binder without sticking to each other. If necessary, the stored gels can easily be scanned with a densitometer at any time. ACKNOWLEDGMENTS I thank Mark Zukowski, Barbara Karan, and Evryll Swanson for help during the preparation of this manuscript and Joan Bennett for typing the manuscript.
REFERENCES 1. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 2. O’Farrell, P. Z., Goodman, H. M., and O’Farrell, P. H. (1977) Cell 12, 1133-I 142. 3. Hoffman, W. L., and Dowben, R. M. (1978) Anal. Biochem. 89,540-549. 4. Popescu, 0. (1983) Electrophoresis 4,432-433. 5. Juang, R.-H., Chang, Y.-D., Sung, H.-Y., and Su, J.-C. (1984) Anal. Biochem. 141,348-350.