Question

You isolate a subcellular fraction and find the protein concentration to be 40 mg/mL.

a) Show all calculations and explain how you would prepare 50 µL of a 4 mg/mL final concentration after dilution in 2X sample buffer. (Final concentration after mixing with 2X sample should be 4 mg/mL).

b) You are asked to load 20 µg total proteins into a well in the polyacrylamide gel. How many microlitres of the sample prepared above should you load onto the gel?

Answer 1

a) stock concentration(M1)= 40 mg/mL

final concentration required (M2)= 4 mg/mL

final volume(V2)=50 uL

volume of stock requred (V1)= ?

M1V1=M2V2

V1=M2V2/M1

= (450)/40

= 5 uL

take 5 uL of the stock solution and add 45 uL of 2X sample buffer to it to make the final concentration=4 mg/mL

b) amount of protein required= 20 ug

concentration of the protein= 4 mg/mL

1 mg= 1000 ug

1 mL= 1000 uL

so 1 mg/mL= 1 ug/uL

so 4 mg/mL= 4 ug/uL

concentration of the protein= 4 ug/uL

In 1 uL there is 4 ug of protein, we need 20 ug, so volume of the solution required= amount of protein required/concentration of protein= 20 ug/4 ug/uL=5 uL

volume of the diluted sample to be taken= 5 uL