Microplate Bradford assay - very urgent (Nov/16/2009 )

Dear all,


I have small quantity of a protein, and I want to do microplate Bradfor dassay, can anybody explain to me how to set up the BSA standard, and the precise amount in every well, and do I need to dilute my sample or not?

Hi rimal
this might help..
if you have stil furtehr questions.. please be speific and we might be able to help you!!

Hi!! Rimal ,
When you are doing bradford's assay on a microplate, your sample should be about 40 microliters and reagent 200 microliters. You can prepare your BSA standard should start from 0.5 micrograms to 10 micrograms. You must do a trial run only with the standard curve first. Normally on a microplate you acheive saturation at about 8 micrograms. The highest protein concentration you take should not be more than 5 micrograms. Detergents like NP 40, TritonX 100, Buffers like Tris and HEPES also interfere with the assay. So be carefull to make your standard in buffer that you use. Also use the buffer for blank.

rimal on Nov 17 2009, 10:00 AM said:

Thank you very much. I have purified x protein, and I don't know its concentration, also the amount is very small. I have Bradford reagent from Sigma but I couldn't understand their instruction of use, especially how to chose the range of SBA standard. Their range for microplate is 0.1-1.4 mg/ml and I am afraid my protein conc. may be less? so if possible I need further explanation to their protocol.

http://www.sigmaaldrich.com/etc/medialib/d...mp/b6916bul.pdf

WELL ACCORDING TO THE INSERT, THE MINIMUM THE PRODUCT (BRADFORD-SIGMA) CAN DETECT IS 0.1MG/ML

I fear you cant do much about it!!!
The best that i can suggest is try preparing the dilutions in your desired range even if it is lower than 0.1mg/ml and instead of taking the 250 mcl bradford that they prescribe take 300 ,cl and take 10 mcl of standard sample (make dilutions of sample for accuracy).
In short, use the sigma product and follow the pierce protocol that i have provided and hope for the best.
Best Luck!!!

Make concentration 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 and 0.45. Add 10 ul in quadruplicate, add 200 Dye and there you go. Dilute the sample so that it falls within the standard curve (E.g 1/20, 1/40 and 1/80)

Hi all, I have some questions regarding Microplate Bradford assay. Guess I could just reply in this thread instead of creating a new one.
I insert in my E.Coli bacteria a high copy plasmid (copy no. of about 20-50) containing inducible gene. The gene encodes a protein to be secreted out by the bacteria. I'd like to roughly monitor the concentration of this protein (upon induction) in the extracellular solution. However, I am not sure whether Bradford assay (or other type of total protein concentration assay) is suitable for this purpose. My concern is whether the concentration of this protein is significant compared to that of all other proteins always present in extracellular solution.
Any thought would be really appreciated.

G_B

Hi GB.... as u correctly said.. a total protein assay will not help u much in quantifying your protein of interest... i can suggest two things... one is quantitate total protein thru bradford or any other assay and run SDS-PAGE/WB in replicas and calculate the percent of your protein... which may be a bit more approximate
secondly the best practice is to do an ELISA for your protein of interest!!!

Best luck!!

on second thoughts you can also use a hplc to quantitate your protein of interest... but generally in harvest samples getting tat kind of resolutions is not that easy... so i will still vouch for ELISA!!!

Hi, thank you Pradeep!

My sup told me to avoid using antibody as it might take time. So I guess I'll try Bradford with SDS..