Question

You are preparing a primary antibody solution [a 1:5,000 dilution of anti-NADPH oxidase in 5% milk, TBST] for doing western blots. There are 5 total blots, each blot with dimensions of 8.6 cm x 6.7 cm. You want to prepare a sufficient volume of antibody solution such that each blot can be covered with antibody to a depth of 1.2 mm. You figure that you will need to make 10% extra volume of the bulk antibody solution to enable you to comfortably make 5 aliquots of equal volume of antibody solution. Assume you have sufficient volumes of stock anti-NADPH oxidase antibody and 5% milk, TBST. How would you make the bulk antibody solution? Show all calculations and write an explanation of your thinking.

Answer 1

Answer - You need to add 7.6 ul of anti-NADPH oxidase antibody in 38 ml of 5 % milk,TBST ( total volume = 38.76 ml ) .Further can be divided for 5 blots = 38.76/5 = approx 7.7 ml of 5 % milk,TBST added antibody in each blot.

Explanation

Volume for 1 blot = lengthX width X height = 8.6 cm X 6.7 cm X1.2 mm (0.12 cm ) = 6.9144 cm3  

1 ml = 1 cm3

Hence , one blot volume is 6.9144 ml. So, for the 5 blots the total volume would be = 6.9144 X 5 = 34.572 ml of the 5 % milk TBST is required.

As suggested you need to make 10% extra volume of the bulk antibody solution to enable you to comfortably make 5 aliquots of equal volume of antibody solution.

extra amount of antibody solution needed =34.572 ml X 10 % = 3.45 ml

Thus, the bulk antibody solution volume =34.572 ml +3.45 ml = 38.022 ml ( for 5 aliquotes)

Now comes to the dilution of the anti-NADPH oxidase primary antibody  in 5% milk,TBST for doing western blots =1:5000 (it means that for 5000ul or 5 ml of the 5% milk,TBSTwill need 1 ul of the antibody )

For 38.022 ml of 5% milk,TBST will need the anti-NADPH oxidase primary antibody = 1ul /5 ml X 38.022 ml =7.6044 ml or 7.6 ul of antibody .