AES Application Focus Two-dimensional Gel Electrophoresis Page 1 Two-Dimensional Gel Electrophoresis Links GE: www.gehealthcare.com Bio-Rad: www.bio-rad.com CBS Scientific: www.cbssci.com Kendrick Labs: www.kendricklabs.com Syngene: www.syngene.com Adapted from Chapter 7, Gel Electrophoresis of Proteins, by David E. Garfin, Pages 197-268, in Essential Cell Biology, Volume 1: Cell Structure, A Practical Approach, Edited by John Davey and Mike Lord, Oxford University Press, Oxford UK (2003). Used by permission of Oxford University Press. 2-D PAGE provides the highest resolution separation method for proteins (1, 2). Following a first dimension IEF, proteins are subjected to SDS-PAGE in a perpendicular direction (see the Applications Focuses on Isoelectric Focusing and Gel Electrophoresis of Proteins on this website). The technique is a true orthogonal procedure in that the two separation mechanisms are based on different physical principles (they are orthogonal in that sense) and the two separations are done at right angles to one another (they are geometrically orthogonal). Thousands of polypeptides can be resolved in a single 2-D PAGE slab gel. The technique works best with soluble proteins such as those from serum or cytoplasm. It is relatively labor intensive for an electrophoresis technique, requiring a relatively high skill level for best results. The best approach for 2-D PAGE is to run the IEF first dimension using IPG strips, and the best approach to obtaining IPG strips is to purchase them already made. Following IEF, an IPG strip is inserted into the gel cassette on top of the SDS-PAGE slab gel. The SDS-PAGE gel is run and stained as with one-dimensional electrophoresis. The difference between 1-D PAGE and 2-D PAGE gels is that the protein patterns in 2-D PAGE are spots rather than bands (Figure 1).
AES Application Focus Two-dimensional Gel Electrophoresis Page 2 Figure 1. Two-dimensional polyacrylamide gel electrophoresis. Proteins from a lysate of Escherichia coli were subjected to IEF in a 17-cm IPG strip spanning the ph range of 4-to-7. The strip containing focused proteins was transferred to an 18 x 20 cm SDS-PAGE gel (8-16%T) and subjected to electrophoresis. Proteins in the gel were stained with SYPRO Ruby Gel Stain and the image shown was captured with a laser-based instrument. A second IPG strip was run in parallel and stained with colloidal CBB G-250. Its image is superimposed above that of the 2-D PAGE gel for comparison.
AES Application Focus Two-dimensional Gel Electrophoresis Page 3 Protocol 1. Two-dimensional polyacrylamide gel electrophoresis Equipment and reagents Isoelectric focusing apparatus appropriate for use with IPGs Electrophoresis cell Power supply IPG trays or disposable 2-ml and 5-ml pipettes for treating IPG strips. IPG strips in suitable ph range and length to fit the second dimension gel. SDS-PAGE gel: commercial precast or made according to Protocol 2 in the Application Focus on Gel electrophoresis of Proteins on this website. The well in the gel must match the length of the IPG strip. IPG sample solution a IPG equilibration solution b 0.5% agarose in Laemmli SDS-PAGE electrode buffer c Method 1. Mix the sample proteins with IPG sample solution so that the proteins are at a final concentration of about 0.1-1 mg/ml, depending on the staining method to be used. The ph range of the carrier ampholytes in the sample solution should match the ph of the IPG strip. Concentrations of carrier ampholytes exceeding 0.2% (w/v) result in extended focusing times. 2. Carefully peel the protective plastic sheet from the (dehydrated) IPG strip. Rehydrate the IPG in protein solution (step 1) with the gel facing down in a tray or 2-ml disposable pipette. See the recommendations of the manufacturer of the IPG strip for the correct volume of solution to use in order to rehydrate the IPG strip properly. As a guide, 125 µl fully rehydrates a 7-cm strip, 200 µl rehydrates an 11-cm strip, and 300 µl rehydrates a 17-cm strip. After one hour in a tray, cover the strip with light silicon oil. If a pipette is used rather than a tray, close off both of its ends with Parafilm. Allow at least 12 hours for thorough rehydration. Use a fine-point forceps for all manipulations of IPG strips. 3. Transfer the IPG strip to the IEF cell and carry out the IEF according to the instructions provided by the manufacturer of the cell. The strip must be covered with light silicon oil during focusing. It might take some trial and error to arrive at proper focusing conditions for each different protein sample. Often 20-30 kvh (kilovolt hours) are sufficient for 7-cm IPG strips, 40-50 kvh for 11-cm strips and 60-70 kvh for 17-cm strips. 4. Thaw one 10-ml aliquot of IPG protein equilibration solution for each IPG strip. Make sure that all components of the solution are thoroughly dissolved. Gentle warming may be required. Divide the aliquot into two 5-ml portions. To one of the 5-ml portions, add 50 mg of dithiothreitol, DTT (to 1%, or 65 mm). Add 75 mg of iodoacetamide, IAA, to the other 5-ml portion (to 1.5% or 80 mm). Carry out this step while the IEF run is still in progress so that the IPG strip will not be subjected to drying out.
AES Application Focus Two-dimensional Gel Electrophoresis Page 4 5. After IEF, place the strip with the plastic side down on a damp piece of filter paper and gently blot excess oil and other liquid from the gel with a piece of damp filter paper. 6. Transfer the IPG strip to a tray or 5-ml disposable pipette and incubate it with the 5- ml DTT solution prepared in Step 4 for 15 min at room temperature with gentle rocking. This reduces disulfide bonds in the proteins in the IPG strip. 7. Remove the IPG strip from the reduction solution and blot off excess liquid as in Step 5. Transfer the strip to the 5-ml IAA solution prepared in Step 4 in a clean trough in a tray or a fresh pipette. Incubate the strip for 15 min at room temperature with gentle rocking to alkylate free sulfhydryl groups in the proteins in the IPG strip. 8. Remove the IPG strip from the alkylation solution and blot off excess liquid as in Step 5. Transfer the strip to the top of the gel cassette holding the second dimension gel. Use the forceps to place the plastic backing of the strip on the back plate of the gel cassette. Use a spatula to push the strip into the cassette above the gel so that it is within 1 mm of the bottom of the well. 9. Melt the agarose and let it cool until it is warm to the touch but still molten. Pipette the molten agarose into the gel cassette to seal the IPG strip to the gel. Hold the cassette at a slight angle as the agarose is being pipetted into it to provide a way for bubbles to escape. There must not be any bubbles trapped in the thin agarose layer between the IPG strip and the top of the gel. It may take some practice to become proficient in layering the agarose. 10. When the agarose has hardened, scrape excess agarose from the top of the cassette and transfer the cassette to the electrophoresis cell. Run the second dimension as for standard SDS-PAGE. Stain the completed gel as for SDS-PAGE (see the Applications Focuses on Gel Electrophoresis of Proteins and Detection of Proteins in Gels on this website). a IPG sample solution is 8 M urea, 2% (w/v) CHAPS, 0.3% (w/v) dithiothreitol (DTT) (20 mm), 0.2% (w/v) carrier ampholytes. Dissolve 48 g of urea, 2 g of CHAPS, 0.3 g of DTT, and 0.5 ml of carrier ampholytes (assumed to be at 40% w/v) in 60 ml of water. Adjust the final volume to 100 ml if necessary. Aliquot and freeze 5 ml portions. Mix this solution well upon thawing it for use so that all of the urea and CHAPS dissolve. Gentle warming may be required. b IPG equilibration solution is 6 M urea, 75 mm Tris-Cl, ph 8.8, 2% (w/v) SDS. Dissolve 36 g of urea, 5 ml of 1.5 M Tris-Cl, ph 8.8 (see Protocol 2 in the Application Focus on Gel Electrophoresis of Proteins on this website), 2 g of SDS in 60 ml of water. Adjust the final volume to 100 ml. Aliquot and freeze 10-ml portions. Mix this solution well upon thawing it for use so that all of the urea and SDS dissolve. c To prepare 0.5% agarose solution, add 0.5 g of low melting point agarose to 100 ml of Laemmli SDS-PAGE electrode buffer (see Table 2 in the Application Focus on Gel Electrophoresis of Proteins on this website). Add a spatula-tip amount of bromophenol blue to impart color. Melt the agarose on a hot plate or in a microwave oven. Mix the solution well and store the resultant gel at room temperature. The agarose must be remelted before each use.
AES Application Focus Two-dimensional Gel Electrophoresis Page 5 A large part of the success of a 2-D PAGE run is determined by careful sample preparation. This topic and several of the nuances of 2-D PAGE are outside the scope of this Application Focus. Those interested should consult References 1-8. References 1. Harrington, M.G., Gudeman, D., Zewert, T., Yun, M., and Hood, L. (1991). In Methods: a companion to Methods in enzymology (ed. M.G. Harrington). Vol. 3, No. 2, p. 98. Academic Press, San Diego. 2. Herbert, B.R., Sanchez, J.-C., and Bini, L. (1997). In Proteome research: new frontiers in functional genomics (ed. M.R. Wilkins, K.L. Williams, R.D. Appel, and D.F. Hochstrasser), p. 13. Springer, Berlin. 3. Sanchez, J.-C., Rouge, V., Pisteur, M., Ravier, F., Tonella, L., Moosmayer, M. Wilkins, M.R., and Hochstrasser, D.F. (1997). Electrophoresis, 18, 324. 4. Hanash, S.M. (1998). In Gel electrophoresis of proteins: a practical approach (3rd edn) (ed. B.D. Hames), p. 189. Oxford University Press, Oxford. 5. Link, A.J. (ed.) (1999). 2-D proteome analysis protocols. Human Press, Totowa, NJ. 6. Rabilloud, T. (ed.) (2000). Proteome research: two-dimensional gel electrophoresis and identification methods. Springer, Berlin. 7. Rabilloud, T. (1996). Electrophoresis, 17, 813. 8. Molloy, M.P. (2000). Anal. Biochem., 280, 1.