# protein concentration - calculation question (Aug/10/2008 )

Hello all,

I come to you with a simple question, I hope I get an answer. I extracted and purified protein from a cell culture and the quantified my protein concentration by Bradford assay. Here is how I did it:
1. pipette in a 96 well plate 2.5, 5, 7.5 and 10 microliters of 1mg/ml BSA (triplicate).
2. take to 160 microliters with water, that is 147.5, 145, 142.5 and 150 microliters of water, and of course a blank.
4. repeat for unknowns using 2 ul of each sample.
5. read OD @ 595 nm.
6. plot and solve the equation for each sample.
7. Since I loaded 2 ul of the sample, I simply divided the result by 2, getting the amount of protein in 1 ul of sample. I never used a dilution factor in my calculations.

I'm not sure if my procedure for the last step is correct. If it is not, would somebody be kind enough to point me to the right direction?

Cheers.

-Pancho-

QUOTE (Pancho @ Aug 10 2008, 04:27 PM)
Hello all,

I come to you with a simple question, I hope I get an answer. I extracted and purified protein from a cell culture and the quantified my protein concentration by Bradford assay. Here is how I did it:
1. pipette in a 96 well plate 2.5, 5, 7.5 and 10 microliters of 1mg/ml BSA (triplicate).
2. take to 160 microliters with water, that is 147.5, 145, 142.5 and 150 microliters of water, and of course a blank.
4. repeat for unknowns using 2 ul of each sample.
5. read OD @ 595 nm.
6. plot and solve the equation for each sample.
7. Since I loaded 2 ul of the sample, I simply divided the result by 2, getting the amount of protein in 1 ul of sample. I never used a dilution factor in my calculations.

I'm not sure if my procedure for the last step is correct. If it is not, would somebody be kind enough to point me to the right direction?

Cheers.

Since you use 2 ul for each unknown samples, if I were you, I will do:

1. pipette in a 96 well plate 2 microliters of 0, 1, 2, 4, 6, 10 mg/ml BSA (triplicate).
2. take to 158 microliters with water, and of course a blank.