SDS-Polyacrylamide Gel Electrophoresis

Denatured using SDS in gel and buffer

1. Making the gel:
4% Stacking gel preparation:

        Deionized H2O                               6.1 ml
        Tris-HCl (0.5 M), pH6.8                 2.5 ml
        10% SDS (w/v)                             100 ul
        Acrylamide/bis (30% stock)         1.33 ml 
        or use 40% acrylamide                  (1.0 ml)
        10% APS (fresh)                            50 ul   (f.c.= 0.05%)
        TEMED                                           10 ul   (f.c.=0.1%)
                                                                                                

Separating gel preparation:     12%                  7.5% 

        Deionized H2O                           3.35 ml                       4.85 ml
        Tris-HCl (1.5M), 30% Acrylamide/Bis-Acrylamide: 
        (per 100. ml)
        Ratio                   Acrylamide      Bis-Acrylamide
        29:1                    29. g                    1.0 g                           
        19:1 (RPA)          28.5 g                  1.5 g

Detection Buffer: 1 M Tris-HCl, pH 9.5, 1 M NaCl.
Tris buffered Saline with Tween 20 (TTBS): 0.1 M Tris HCl, pH 7.5, 0.1 M NaCl 
	plus 0.1% (w/v) Tween 20.

Blocking Buffer: 5% (w/v) Dried powdered milk, 0.1% (v/v) Tween 20.

1X Electrophoresis buffer:
        1.51 g Tris base
        7.20 g glycine
        0.5 g SDS or 5.0 ml of 10% SDS
        H20 up to 400 ml, adjust pH to 8.3 with 1 M NaOH 
        Bring up to 500 ml

1X Transfer buffer (500 ml):
        1.51 g Tris base
        7.20 g glycine
        H20 up to 400 ml, adjust pH to 8.3 with 1 M NaOH 
        Bring up to 500 ml