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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 perpendiculardirection (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 twoseparation 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 aregeometrically 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 serumor 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-DPAGE are spots rather than bands (Figure 1).

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Figure 1.Two-dimensional polyacrylamide gel electrophoresis. Proteins from a lysate ofEscherichia coliwere subjected to IEF in a 17-cm IPG strip spanning the pH range of 4-to-7. Thestrip containing focused proteins was transferred to an 18 x 20 cm SDS-PAGE gel (8-16%T) andsubjected to electrophoresis. Proteins in the gel were stained with SYPRO Ruby Gel Stain andthe 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-DPAGE gel for comparison.

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Protocol 1.Two-dimensional polyacrylamide gel electrophoresis

Equipment and reagents

Isoelectric focusing apparatus appropriatefor 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 ormade according to Protocol 2in theApplication Focus on Gel electrophoresis ofProteins on this website. The well in the gelmust match the length of the IPG strip.

IPG sample solutiona

IPG equilibration solutionb

0.5% agarose in Laemmli SDS-PAGE

electrode bufferc

Method

1. Mix the sample proteins with IPG sample solution so that the proteins are at a finalconcentration 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 inextended 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-mldisposable pipette. See the recommendations of the manufacturer of the IPG strip forthe correct volume of solution to use in order to rehydrate the IPG strip properly. As a

guide, 125 l fully rehydrates a 7-cm strip, 200 l rehydrates an 11-cm strip, and 300

l 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 allmanipulations 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 withlight 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 (kilovolthours) are sufficient for 7-cm IPG strips, 40-50 kVh for 11-cm strips and 60-70 kVhfor 17-cm strips.

4. Thaw one 10-ml aliquot of IPG protein equilibration solution for each IPG strip. Makesure that all components of the solution are thoroughly dissolved. Gentle warmingmay be required. Divide the aliquot into two 5-ml portions. To one of the 5-mlportions, 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 stepwhile the IEF run is still in progress so that the IPG strip will not be subjected to

drying out.

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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 filterpaper.

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 Step5. Transfer the strip to the 5-ml IAA solution prepared in Step 4 in a clean trough in atray 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 gelcassette. 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 thecassette at a slight angle as the agarose is being pipetted into it to provide a way forbubbles 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).

aIPG sample solution is 8 Murea, 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 ofcarrier ampholytes (assumed to be at 40% w/v) in 60 ml of water. Adjust the final volume to 100ml if necessary. Aliquot and freeze 5 ml portions. Mix this solution well upon thawing it for use sothat all of the urea and CHAPS dissolve. Gentle warming may be required.

bIPG equilibration solution is 6 Murea, 75 mMTris-Cl, pH 8.8, 2% (w/v) SDS. Dissolve 36 g ofurea, 5 ml of 1.5 MTris-Cl, pH 8.8 (see Protocol 2in the Application Focus on GelElectrophoresis of Proteins on this website), 2 g of SDS in 60 ml of water. Adjust the finalvolume to 100 ml. Aliquot and freeze 10-ml portions. Mix this solution well upon thawing it foruse so that all of the urea and SDS dissolve.

cTo 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 2in the Application Focus on Gel Electrophoresis ofProteins on this website). Add a spatula-tip amount of bromophenol blue to impart color. Melt theagarose on a hot plate or in a microwaveoven. Mix the solution well and store the resultant gelat room temperature. The agarose must be remelted before each use.

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A large part of the success of a 2-D PAGE run is determined by careful samplepreparation. 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). InMethods: 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). InProteome research: new

frontiers in functional genomics(ed. M.R. Wilkins, K.L. Williams, R.D. Appel, andD.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(3rdedn) (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.