Question

7) If you are making a 1:10 dilution of a 10mg/ml BSA solution using 90ul of water as diluent, how much 10mg/ml of BSA would you add? 8) What would be your final concentration of BSA? 9) If you assayed both these solutions using the Bradford assay which one would have a higher O.D.? 10) What device would you use to measure O.D.? 11) In your assay you would need a blank. What would you use as a blank? Water, 10mg/ml BSA, 5mg/ml BSA, 0.5mg/ml BSA? 12) What would you expect the O.D. of Blank to be?

Answer 1

7) To make a 1: 10 dilution of stock 10 mg/ ml solution. We need to dilute the solution 10 times, which means we need 1 part of the stock BSA solution and 9 part of diluent (water). Therefore, if we add 10 ul of stock BSA solution to 90 ul water we will get 10 times diluted BSA solution of its original concentration. So, 10 ul of 10 mg/ ml BSA solution is required to add in 90 ul of water as diluent.

8) The concentration of the stock BSA solution is 10 mg/ml. This means each ml of solution contains 10 mg of BSA. We added 10 ul of stock BSA solution to get 1:10 dilution. With this information, we can calculate the concentration of the diluted solution in the following manner.

1 ml = 1000 ul

1000 ul = 10 mg ( BSA stock solution)

10 ul = 10 * 10 / 1000

= 0.1 mg ( 1 mg= 1000 ug, so 0.1 mg = 100 ug)

Therefore the concentration of the diluted sample is 0.1 mg.

9) Bradfordford assay is a protein estimation assay which is based on the shift of dye Coomassie brilliant blue G-250. It is an absorbance based colorimetric assay in which dye binds to the protein molecule present in the sample and gives blue color. In the absence of protein, the color remains brown. SO, so the color depends on how on the concentration of protein. So O.D. of 10 mg/ml of BSA will be more than a diluted sample because 10 mg/ml solution has more amount of BSA (protein.)

10) The device spectrophotometer will be used to measure O.D.

11) In the assay, water will be used as a blank because it is used as a medium to prepare a BSA solution and water is not having any added analyte which has to be measured whereas others have BSA.  

12) The O.D. of blank should be zero.