Bradford-protein problem

[bandg]

Dear all,
I face a problem the last weeks I wanted to discuss with u.I extract total protein from cultured cells and I determine the concentration of the lysates by Bradford.It looks that my samples are protein-rich. The problem is that after carrying out the western blot, ponceau as well actin show that the protein amount I supposed to load is much lower.

In few words Bradford shows much protein and western only few. However I use other control proteins and these show that western blot and transfer work well.

I checked the reagents of my lysis buffer and the concentrations are compatible with bradford. So I was wondering what it may go wrong and interferes with bradford and how I could solve the problem.Any idea?

Thanks in advance for any help.

[Spike1985]

It might depend on what you have your protein sample diluted in. I previously had a similar problem only to find that the 1% SDS that i used to dissolve my protein was not compatible with the bradford reagent protocol. I had to determine the protein concentration using the lowry method instead.

[Jyerka]

I f I read this right, you are comparing Bradford with an estimation from Western Blot. I would not quantitate from a western blot, it is very misleading without a well characterized standard curve etc. Even then it is sticky.

[bloo_1]

Originally Posted by Jyerka

I f I read this right, you are comparing Bradford with an estimation from Western Blot. I would not quantitate from a western blot, it is very misleading without a well characterized standard curve etc. Even then it is sticky.

I don't think that was the problem, but rather something similar like mine. It's determining the total protein content of a lysate and basically getting a huge overestimation of protein content. For example, I lysed erythrocytes, bradford gave me that the lysate contains an estimated 300 mg/ml protein (wow, right?), but when I put it on gel there's nothing (e.g. with CBB stain). I was going to blame it on haeme or haemoglobin interference in assay but now I see that this might not be the only case.