SDS-Polyacrylamide Gel Electrophoresis at pH

This system allows the separation of alkali-labile proteins (e.g., acylphosphate phosphoproteins) under denaturating conditions according to their molar mass. Despite the low acrylamide concentration (%T = 5.61, %C = 3.61), the separation force is remarkable. Because it is a SDS-containing system, the migration is from to + despite the low pH. 

B 1 M sodium phosphate-phosphoric acid buffer, pH 2.4 C 20% SDS (w/v) in ddH20 

Mix the gel according to Table 2.7, pour the mixture into the cassette and cover with Soln. 1 (Table 2.7). The polymerization takes place very slowly therefore, the gel should be prepared 36-48 h before electrophoresis. A stacking gel is not needed. 

Dissolve samples in buffer G. Electrode buffer is Soln H. Applied voltage should be 8 -10 V per centimeter of gel length. Tracking dye has to be an anionic one intensively colored at pH 2.4 (e.g., cresol red or Pyronin Y). 

The SDS-free PAGE system described by Panyim and Chalkley is suitable especially for small basic proteins (e.g., histones). Additives such as non-ionic detergents as Triton XlOO [0.05-0.1% (w/v)] may additionally increase the resolution. 

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