SDS-PAGE for 2D

11cm IPG Strips

Materials:

• Urea (Fluka)
• SDS (Bio-Rad)
• Glycerol (Fisher)
• Iodoacetamide (Bio-Rad)
• 1.5M Tris/HCl, pH 8.8 (Bio-Rad)
• Equilibration Buffer (See recipe below)
• Tris/Glycine/SDS Running Buffer, 10X (Bio-Rad)
• 0.5% Overlay Agarose (Bio-Rad 163-2111)
• Criterion 8-16% Tris-Glycine gel, IPG + 1 well format (BioRad 345-0105)
• Precision Plus Protein Standards, unstained (Bio-Rad 161-0363)
• Tri-(2-carboxyethyl) phosphine (TCEP), (Protein-S-S-Reductant, GenoTechnology,Inc)

Reagents and Solutions:

Equilibration Buffer:
• 6M Urea
• 2% SDS
• 50 mM Tris/HCl pH 8.8
• 20% Glycerol

Method:

1. Incubate each IPG strip in 4 ml Equilibration Buffer + 10 μl/ml TCEP. Incubate 15 min at RT on shaker. Drain buffer from strip.

2. Incubate each strip in 4 ml Equilibration Buffer + 25 mg/ml iodoacetamide for 15 min at RT on shaker.

3. Rinse strip in 1X Tris-Glycine-SDS running buffer and place in IPG well of gel with the positive end of the strip on the left side of the gel. With forceps or spatula, push the strip on the plastic backing, pressing it firmly into place against the SDS gel. Check to make sure that there are no air bubbles at this interface.

4. Cover strip with molten 0.5% overlay agarose. Again check for the presence of air bubbles at the strip/gel interface.

5. Add 5 μl unstained molecular weight standards in M.W. well.

6. Place gels in electrophoresis apparatus. Run at 150 V for approximately 2.25 hr at 4°C in prechilled 1X Running buffer.