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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

SDS-PAGE OF IMMOBILIZED pH GRADIENT (IPG) STRIPS FOR THE SECOND DIMENSION

SDS-PAGE of Immobilized pH Gradient (IPG) Strips for the Second Dimension
Contributor: The Electrophoresis Laboratory at Geneva University Hospital
 
Overview
Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is one of the most powerful techniques for the study of protein expression and their post-translational modifications.
 
Procedure
A. Preparation of Acrylamide Gel for Second Dimension

1. Prepare the second dimension resolving gel solution by combining (see Hint #1 and #2):

375 mM Tris-HCl, pH 8.8

Acrylamide/Piperazine Diacrylyl (9 to 16 % T and 2.6 % C)

5 mM Sodium Thiosulfate

0.05 % (v/v) TEMED

0.1 % (v/v) Ammonium Persulfate (APS)

2. Pour the Acrylamide solution into a 16 cm x 20 cm glass plate sandwich with 1.5 mm spacers until solution is approximately 0.7 cm from the top of the glass plates.

3. Overlay the Acrylamide solution with 2-Butanol and allow to polymerize for 2 hours.

4. Remove the overlay of 2-Butanol and replace it with Tris-Glycine-SDS Buffer.

5. Allow gel to sit overnight with Tris-Glycine-SDS Buffer overlay.

6. Do not prepare a stacking gel (see Hint #3).

B. Immobilized pH Gradient (IPG) Gel Strip Transfer

1. Cut the IPG gel strips (already run in the first dimension; see Protocols on Immobilized pH Gradient (IPG) As First Dimension for 2D Protein Gels) to size.

2. Overlay the second dimension gels with Agarose Solution.

3. Immediately load the IPG gel strips through the melted agarose, making sure the strip is centered, that it has sunk down as far as possible in the embedding medium, and that there are no bubbles.

C. Running Conditions

1. Run the gel at 40 mA constant current for 5 hours at 8 to 12°C.

2. Process the gel to stain the proteins (see Protocol on Silver Staining Proteins in Acrylamide Gels or other appropriate protocols).

Solutions
Trailing Buffer   Tris-Glycine-SDS Buffer
Leading Buffer   375 mM Tris-HCl, pH 8.8
Tris-Glycine-SDS Buffer   0.1% (w/v) SDS
pH 8.3
25 mM Tris
198 mM Glycine
Agarose Solution   0.5% (w/v) Agarose
Heat to approximately 70°C
Prepared in Tris-Glycine-SDS Buffer
Use immediately
 
BioReagents and Chemicals
Agarose
APS
Sodium Thiosulfate
Glycine
Tris-HCl
TEMED
SDS
Piperazine Diacrylyl
Acrylamide
2-Butanol
 
Protocol Hints
1. The contributor uses piperazine diacrylyl as the polymerizing agent because it reduces N-teminal protein blockage, gives better protein resolution, and reduces diamine silver staining background.

2. No SDS is included in the gel. This seems to prevent the formation of micelles, which contain acrylamide monomer, thus increasing the homogeneity of pore size and reducing the concentration of unpolymerized monomer in the polyacrylamide. The SDS used in the gel running buffer is sufficient to maintain the necessary negative charge on proteins.

3. The combination of the IPG strip and agarose obviates the need for a stacking gel.