BIOL 1406

PreLab 8b.2

How do I prepare the BSA dilutions needed to create the standard curve for the Bradford assay?


In order to prepare a standard curve for the Bradford assay, you will make 0.1 mL of each of the following BSA dilutions: 0 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1.0 mg/mL, 1.2 mg/mL, and 1.4 mg/mL. To make these dilutions, you will be provided with a stock solution containing 2 mg/mL of BSA.

 

Your Turn
Calculate how many μL of stock BSA (2 mg/mL) and how many μL of dH2O you need to make 0.1 mL of each of the dilutions shown in the table below. Since you will need this information during lab, enter the required amounts in this table and in your lab notebook. For a review of how to do the calculations for parallel dilutions, check Prelab 2.4.
 

Preparation of 0.1 mL of each BSA dilution from a 2 mg/mL stock solution of BSA

Cuvette # Final BSA concentration (mg/mL) Amount of stock BSA needed (μL) Amount of dH2O needed (μL)
1 0.0 Check your answer. Check your answer.
2 0.1 Check your answer. Check your answer.
3 0.2 Check your answer. Check your answer.
4 0.4 Check your answer.

Check your answer.
5 0.6 Check your answer. Check your answer.
6 0.8 Check your answer. Check your answer.
7 1.0 Check your answer. Check your answer.
8 1.2 Check your answer. Check your answer.
9 1.4 Check your answer. Check your answer.

 



After you prepare these 9 BSA dilutions, you will add 3.0 mL of Bradford’s Reagent to each cuvette and measure the A595 values. You will then plot a scatter diagram of your data and use linear regression to determine the standard curve for the Bradford assay. The standard curve will show the exact relationship between protein concentration and A595 values. Using the standard curve, you will calculate the protein concentration of the 8 milk fractions that you saved during your purification steps on Day 1.


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