SDS-PAGE Concentration Calculation Method
When preparing SDS-PAGE gels, the concentration of the gel refers to the total concentration of acrylamide, which determines the pore size of the gel and affects the protein separation efficiency. The higher the concentration, the smaller the pores, suitable for separating low molecular weight proteins; the lower the concentration, the larger the pores, suitable for separating high molecular weight proteins.
Formula for Calculating Gel Concentration
Total gel concentration (T) = acrylamide concentration + crosslinker concentration
Typically, the crosslinker refers to N,N'-methylenebisacrylamide (Bis), which is mixed with acrylamide at a fixed ratio, commonly 1:29 (Bis:acrylamide).
Suppose you want to prepare a 12% polyacrylamide gel. If you are using a 30% acrylamide solution (containing a fixed ratio of acrylamide and Bis), you need to calculate how to achieve the 12% working concentration from the 30% stock solution.
Example
If you want to prepare 10 mL of a 12% SDS-PAGE gel using a 30% acrylamide/crosslinker solution (for example, a 29:1 ratio), you would need:
1. 30% Acrylamide Solution
(Required gel concentration / Stock solution acrylamide concentration) × total volume
(12% / 30%) × 10 mL = 4 mL
2. Deionized Water (to Adjust the Total Volume to 10 mL)
Total volume - volume of acrylamide solution - volume of other components
10 mL - 4 mL - volume of other components
3. Other Components (Tris buffer, SDS, APS, TEMED) Are Added in Fixed Volumes According to the Recipe
If the volumes of other components like Tris-HCl, SDS, TEMED, and APS are 3.75 mL, 0.1 mL, 0.05 mL, and 0.1 mL respectively, then the volume of deionized water should be:
10 mL - 4 mL - 3.75 mL - 0.1 mL - 0.05 mL - 0.1 mL = 2 mL
Therefore, you would need to add 2 mL of deionized water.
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