Bradford Assay - Calculation protein concentration (Jun/30/2008 )
Hi Everyone,
I feel sort of dumb asking this question, but I haven't had much luck finding it anywhere, so here goes:
After lysising cells, I check my protein concetration by Bradford using a microplate reader. I add 200 ul of diluted Bradford reagent to each well of a 96-well plate. I then add BSA to make up my standards (10 ug/ml-40 ug/ml, lower or higher concentrations if necessary). Lastly, I add 1 ul of my samples in duplicate to the wells. Our plate reader calculates concentrations of samples based on the standard curve and the dilution factor. My question is, since I'm adding 1 ul of my sample to 200 ul of Bradford reagent, is my dilution factor 200? And if I added 2 ul of sample instead of 1 ul, would it be a 100X dilution? I'm not sure I'm getting the right protein concentrations when doing my Bradfords. It's all relative, but it would be nice to know how much protein I'm actually loading onto my gels 
Thanks!
I feel sort of dumb asking this question, but I haven't had much luck finding it anywhere, so here goes:
After lysising cells, I check my protein concetration by Bradford using a microplate reader. I add 200 ul of diluted Bradford reagent to each well of a 96-well plate. I then add BSA to make up my standards (10 ug/ml-40 ug/ml, lower or higher concentrations if necessary). Lastly, I add 1 ul of my samples in duplicate to the wells. Our plate reader calculates concentrations of samples based on the standard curve and the dilution factor. My question is, since I'm adding 1 ul of my sample to 200 ul of Bradford reagent, is my dilution factor 200? And if I added 2 ul of sample instead of 1 ul, would it be a 100X dilution? I'm not sure I'm getting the right protein concentrations when doing my Bradfords. It's all relative, but it would be nice to know how much protein I'm actually loading onto my gels
Thanks!
you do not need to work with a dilution factor if you use the same dilution for the BSA standards; you only need the slope of the linear phase of BSA standard row as a factor to calculate protein from extinction
what volume of your standards are you adding?
if you are adding 1ul of each standard and are comparing your samples to the curve you generate from the standards then you are not diluting your samples at all.
I should clarify:
To make my standard curve, I add 1 ul, 2 ul, 3 ul, 4 ul, etc. of 1 mg/ml BSA directly into the well with 200ul Bradford reagent. I don't dilute it first and then add it to the well. I should probably be diluting my BSA in lysis buffer (!) and be adding equal volumes to each well, but that's another point of discussion.
To make my standard curve, I add 1 ul, 2 ul, 3 ul, 4 ul, etc. of 1 mg/ml BSA directly into the well with 200ul Bradford reagent. I don't dilute it first and then add it to the well. I should probably be diluting my BSA in lysis buffer (!) and be adding equal volumes to each well, but that's another point of discussion.
Just do a mass balance (instead of thinking about concentrations).
Plot abosrbance vs ug BSA in standards. Fit linear equation. Using the raw absorbance for each sample, solve the equation for ug BSA given its absorbance value. Now to get concentration of your sample just divide by the volume of the sample.
Our microplate reader reads the standard curve as a concentration to calculate the protein concentrations. This isn't a problem for me if I do a more traditional Bradford. I'm just wondering if a dilution factor applies to the plate reader the way I'm using it.
To make my standard curve, I add 1 ul, 2 ul, 3 ul, 4 ul, etc. of 1 mg/ml BSA directly into the well with 200ul Bradford reagent. I don't dilute it first and then add it to the well. I should probably be diluting my BSA in lysis buffer (!) and be adding equal volumes to each well, but that's another point of discussion.
Just do a mass balance (instead of thinking about concentrations).
Plot abosrbance vs ug BSA in standards. Fit linear equation. Using the raw absorbance for each sample, solve the equation for ug BSA given its absorbance value. Now to get concentration of your sample just divide by the volume of the sample.
1. You should use a minimal volume of 5 ul for both standards and samples, because 1 ul is very difficult to pipet accurately
2. All standards and all samples should have the same volume (eg 10 ul)
3. Pipet the samples and standards on the plate first, then add the Bradford reagent with a multichannel pipet
1ul+200ul is 201× dilution, which is roughly 200×
2ul+200ul is 101× dilution, which is roughly 100×
I have an additional question about Bradford assays. The chairman of my committee insists that BSA binds about 2X more Bradford reagent than most other proteins and therefore using BSA as your protein standard does not give you a correct quantification of an unknown, in fact you are calculating your sample concentrations at approximately half of what they really are. Has anyone ever heard of this? He says that the standard should be some generic IgG of known concentration. I'm thinking of running the two standards side-by-side and seeing what (if any) differences in OD595 there are.
It is known that different proteins bind the Bradford reagent with different affinities. The ideal standard is made up of the protein you are measuring. Since it is usually not possible, all the results will be based on an approximation. You can get around it by simply expressing your concentrations as BSA-equivalent 
or calibrating your protein vs BSA once, and always calculate it back according to the calibration curve.
a couple of comments.
the dilution caused by the protein determination reagent (bradford, lowry, biuret, etc) is normally not considered when calculating protein concentration. dilution is considered when preparing the sample prior to addition of the reagent. and, best practice is to prepare all samples and standards to the same volume so that reagent is not diluted differently for any sample.
bsa does indeed give ~2x the reading of gamma globulin with the bradford reagent. biorad has a table showing various protein responses with bradford. we determined, by using a few different methods, that gamma globulin was the best general protein standard, with the bradford, for the proteins with which we work. you should do the same for yours.