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SDS PAGE Gels—2nd Dimension Electrophoresis Preparation/Casting 2D Gels: For Preparation of 1 or 12 gels. (Gels 20 cm x 20 cm and 1.5 mm thick) The following recipe is for a 12.5% polyacrylamide gel (12.5%T, 3.33%C) Component 1 gel 12 gels       Acrylamide: bisacrylamide (30%: 1%) 40.25 ml 483 ml 1.5 M Tris HCl, pH 8.8 (4X buffer) 24.8 ml 297.60 mL MilliQ water 33.73 ml 404.74 mL 10% SDS 0.99 ml 11.90 mL TEMED 0.066 ml* 0.794 mL* 10% ammonium persulfate (APS) 0.331 ml* 3.97 mL* Total Volume 100.17 ml 1197.24 mL All components except TEMED and 10% APS are added to a large filtering flask and stirred under vacuum for 15 mins to degas the solution. Add TEMED and APS and gently stir the flask to mix; be careful to not generate bubbles. Immediately pour very gently into gel cassettes. Each gel is immediately overlain with 2-3 ml n-butanol (saturated with water) to create a flat gel surface. Equilibration of IPG gel strips: IPG strips are prepared and equilbrated as previously described. Transfer of equilibrated IPG strips to 2nd dimension gel: After equilibration, the strips are rinsed with MilliQ water, lightly blotted and placed on the 2nd dimension gel. IPG strips are sealed on the 2nd dimension gel with agarose sealing solution. Agarose sealing solution   Component Amount     SDS running buffer (1.5 M Tris-HCl, 10X) 10 ml bromophenol blue 2 mg MilliQ water 90 ml Agarose (low melting) 0.5 g Final Volume 100 ml All ingredients are placed in an erlenmeyer flask (500ml) and stirred with heating until the agarose is completely dissolved. Cool the agarose solution to ca. 65°C. Dispense ca. 1.5-2 ml agarose solution over the IPG strip and allow agarose to solidify. Excess agarose solution can be stored at room temperature and used at a later date. Running 2nd Dimension The gels are placed in the 2nd dimension tank (Dodeca Cell, BioRad) filled with 2.3 L Tris/ glycine/SDS (10X) running buffer and 20.7 L MilliQ water . Electrophoresis running buffer is circulated at 4°C.Gels are run at 90 V for ca. 16 hours. The voltage is then increased to 120-130 V for 2-3 hours until the bromophenol blue tracking dye is 1-2 cm from the bottom of the gel. Top of page   Protocols The Michigan Proteome Consortium follows a standard set of protocols listed below: Whole cell lysate Membrane proteins IPG 1st dimension SDS page Colloidal coomassie stain Fluorescence stain Silver stain In-gel digestion References and suggested reading

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