# 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:

Thanks!

-montana-

QUOTE (montana @ Jun 30 2008, 11:32 AM)
Hi Everyone,

I feel sort of dumb asking this question, but I haven't had much luck finding it anywhere, so here goes:

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

-The Bearer-

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.

-mdfenko-

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.

-montana-

QUOTE (montana @ Jun 30 2008, 04:57 PM)
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.

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.

-transient-

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.

QUOTE (transient @ Jun 30 2008, 08:28 PM)
QUOTE (montana @ Jun 30 2008, 04:57 PM)
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.

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.

-montana-

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×

-Kupac-

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.

-rkay447-

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.

-Kupac-