Hi Erel Hatan,

I would suggest even try 1 to 5 or 1 to 10 dilution for the test. That is current practice in our group. Would not dilute the whole sample (I don't know for which applications it will be used in your experiments), but just a part of it.

We usually take 10µl of the sample and add 40 or 90µl of water, to achieve the desired dilution.

We place 10µl of the diluted sample per well in a 96 well plate and add 200µl of the BCA reagent. Make also a duplicate or triplicate of your sample, to ensure the repeatability of the assay.

Hope that helps.

Best Regards,

Dmitry

Thank you!

Why do we need to dilute it anyway? In bradford I didnt dilute it

Use 1X PBS for diluting your samples as well as while preparing your BSA standards. Generally 1:5 dilution is followed in case of low concentrated protein samples. BCA is more sensitive than Bradford.

I dont agree with you..i changed my assay from bradford to BCA because the bradford is more sensitive to detergants hence require percipitation..

Sensitivity that I mentioned was with respect to quantification of low concentration of proteins and not wrt detergents.

ok Sheethal Galande I agree with that :-)

so you suggest to dilute my protein :1, :5 and :10 and check to reading in BCA assay?

my experiment is to compare the total protein content in leaves samples treated with herbicide and treated with H2O (control) samples, so in that case if ill treat all the samples with the same dilutions it doesn't matter as long as I get a good Abs rate?

First check with 1:5 if you get absorbance between 0.2 to 0.8 then use that dilution otherwise increase or decrease the dilution for the sample to bring it in that above mentioned range. Undiluted sample will definitely give a high OD. Do write back if your experiment works out. Good luck👍