Question

You have a protein of an unknown concentration but you know that the range is 0.2 mg/mL to 2 mg/mL. You know that typically amounts around 10 µg give a good result on an SDS-PAGE gel. You have 9 wells (1 ladder and 9 others). Using 5 wells, how would you design an experiment to look at the effect of amount of protein loaded on the band appearance on the gel? Remember that you are mixing 10 µL of sample buffer with 30 µL of protein sample. You will need to make 5 different dilutions of the original protein solution. How will you do that?

Answer 1

It is given that around 10 ug of protein should be there in a well for good SDS results.

It is also given that conc of original solution is between 0.2 to 2 mg/ml or 0.2 to 2 ug/ul

Assuming an average value, we consider the original solution to have conc of about 1 ug/ul.

Now, we need 30 ul of protein sample, but if take 30 ul of this original solution, it will give around 30 ug protein, which is way beyond the recommended value of 10 ug protein.

So we prepare the following five different solutions:

(1) Take 10 uL of original protein solution and add 20 uL of distilled water. This will give a 3 times dilution so the resulting conc will be around 0.3 ug/ul, and hence the well will contain around 0.3*30 = 9 ug protein.

(2) Take 6 uL of original protein solution and add 24 uL of distilled water. This will give a 5 times dilution so the resulting conc will be around 0.2 ug/ul, and hence the well will contain around 0.2*30 = 6 ug protein.

(3) Take 12 uL of original protein solution and add 18 uL of distilled water. This will give a 2.5 times dilution so the resulting conc will be around 0.4 ug/ul, and hence the well will contain around 0.4*30 = 12 ug protein.

(1) Take 3 uL of original protein solution and add 27 uL of distilled water. This will give a 10 times dilution so the resulting conc will be around 0.1 ug/ul, and hence the well will contain around 0.1*30 = 3 ug protein.

(1) Take 15 uL of original protein solution and add 15 uL of distilled water. This will give a 2 times dilution so the resulting conc will be around 0.5 ug/ul, and hence the well will contain around 0.5*30 = 15 ug protein.

In this way we will have different amounts of protein content in the five wells, namely 3 ug, 6 ug, 9 ug, 12 ug, 15 ug. This will help us in determining the effect this has on band appearance.