Question

The following is the recipe for 4X sample loading buffer:

1. 4X stacking gel buffer-10 mL

2. 10% SDS- 18 mL

3. beta-mercaptoethanol- 2 mL

4. glycerol- 20 mL

5. Dash bromophenol blue (assume no volume change)

Questions:

1. What is the final concentration of SDS in the solution of 4X sample loading buffer?

2. What is the purpose of the following reagents in 4X sample loading buffer: a) SDS? b) Beta-mercaptoethanol? c) glycerol? d) Bromophenol blue?

Answer 1

1) We begin with 10% SDS 18 mL and at the end of the preparation we obtain 50 mL of the total buffer:

So we can use the dilution factor:

2) SDS is a detergent that breaks the tertiary structure of proteins (so they all finish as linear molecules) and gives them a negative charge along the molecule (so the charge is now proportional to the molecule weight). Beta-mercaptoethanol is a reducing agent that breaks disulfide bonds, this allows the breakage of tertiary and cuaternary structure of proteins. Glycerol makes more viscous the gel to avoid diffusing out of the well. Bromophenol blue is a migration front indicator.