Microplate Bradford with Tissue Whole Cell lysate - (Apr/11/2007 )

Hello,

We've having some issues regarding high bkg in the Bradford and good lysate #. We use a lysis buffer provided in a kit from Actice Motiff. I'm sure the detergent in the lysis buffer is responsibile. The cell lysates we prepare from mouse cochlea have a total volume of ~ 60-80ul/sample. So we don't want waste anymore than we have to. We can't dilute out our lysates to less than 1ug/ul because that makes it to low to use in our ELISA. But to avoid the detergent interference it should be probably diluted much lower. So we possibly might be able to have our lysate in about 160 ul but not much more.

We use The Sigma Bradford reagent. The protocol says to use 5 ul of sample or BSA Std & then 250 ul of Bradford reagent.

Can the Microplate procedure use a different volume of sample and Reagent than 5 ul & 250ul? We'd like to be able to say take the 5ul sample dilute it in water in the microplate with 5ul -? mix then add ? amount Bradford Reagent.

You may consider using BCA method in future, which is less sensitive to detergent, good linear curve vs protein concentration. It does take longer to do the assay and is soemwhat more sensitive to EDTA than braford method.
To answer your question, yes. Its flexible. The amount will be determined by the protein concentration in the samples, such that the reading will fall within the linear range in the standard curve. You may establish a curve with BSA diluted in the same lysis buffer that you use for sample prep. As detergent has an effect on Braford assay method, try to keep the total concentration of lysis buffer the lowest possible and equal in all samples. Good luck.