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.
3. add 40 ul of bio-rad Bradford reagent (5x concentration)
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?

Thank you in advance for taking the time to read and/or answer.

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.
3. add 40 ul of bio-rad Bradford reagent (5x concentration)
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?

Thank you in advance for taking the time to read and/or answer.

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.
3. add 40 ul of bio-rad Bradford reagent (5x concentration)
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 you loaded 2 ul of the sample, you simply compare the conc of your samples with the standard samples.

-NTH-

Yes and no. Your primary problem is that your dilution of the standards is not the same. Technically you want to add the same volume of standard and sample to the bradford reagent. Hence NTH's reply that it would be better to add 2ul of different concentrations of BSA to create the standard curve.

However, I do my bradfords as you described, using different volumes of 2mg/ml BSA to create the standard curve. I understand that because there are different dilutions, my standard curve is not absolutely perfect but this has always given me accurate enough concentrations of my unknown samples to get equal loading. It just depends on how accurate you need to know your concentrations. For equal loading you are looking more for relative concentrations rather than absolute, which the "quick and dirty" method will give. Just understand what you are doing is considered a "quick and dirty" method, why and what needs to be done for the "correct" method. Otherwise, yes, you can divide your calculated concentrations in half and this will give you a fairly good idea of the per ul concentration.

-rkay447-