Hi,

have you tried to filter the stock solution? Is it fresh and stored at 4° C?

Was this solution prepared with MilliQ water?

Have you prepared your BSA solution or it comes from a dealer?

Check your reagents, because Bradford is easily affected by contaminants.

Try new solutions

What is the color of the precipitate? brown or blue?. Obviously, something is wrong. Control pH

Mate check 3 things:

1) A lab made Bradford reagent is tricky to make. Add orthophosphoric acid at the correct time and in correct concentration.

2) Making a perfect BSA stock also requires caution. Try lukewarm distilled water and add BSA by placing the beaker on a rocker-shaker. Avoid formation of froth.

3) Third and the most important point: What is your sample? If there are intact cells present in the samples by any means, they would give precipitates such as you have mentioned.

Hope this info helps.

I too work on proteins and we know how it can be a tricky business

Anyway hope to hear from you weather you get it through.

Hi there again,

thank you very much for all of your contributions, but unfortunately

all your suggestions we already tried.

5xB solution 4deg stored in dark did precipitate (blue precipitates were formed) BSA (stock in MQ water in conc. 1.51ug/ul and stored at -20deg) and with 7 month old

solution it happened first time and never before. It happened also with fresh stock solution. Both of them were filtered through filter paper as well as 0.45um filter.

The pH of it was ~ 1.

I never ever had such a pain with this as I have it now. No obvious reason for such failure. As I wrote before, precipitates are formed already in final konc of BSA 3ug/ml in 1xB solution almost immediately. I have tried to add BSA or cell lysates,... into 5xB and diluted by MQ (as I do usually) as well into 1xB freshly diluted (which is less sensitive, calibration is only 0.98 compare to the first one - 0.999 with almost identical linear fitting parameters over half year).

Anyhow, thank you all for suggestions, hopefully sb comes with sth I did not try to fix this. ;)

Dear a Andrei,

thank's for a tip, I know that instruction manual also that one of Bio Rad,

I use to use them, but unfortunately there is nothing to help me. :(

...take it this way, I did all possibly imaginable and no help, that's why

I asked such a trivial question here, because I'm hopeless :). No obvious

reasons for this situation ;)

Dear Hudec,

I think you should again prepare the reagent and try to dissolve Coommasie Blue G-250 in methanol and then add orthophosphoric acid and water to it. Even when I had made the reagent there was no complete dissolution of the dye so you need to filter the complete reagent and then perform the assay. Try this recipie:

Bradford Reagent (5X concentrate)

100 mg Coomassie Brilliant Blue G-250

47 ml Methanol (100%)

100 ml Phosphoric Acid (85%)

make to 200 ml with double distilled water

Prepare standard curve with BSA Fraction V (1 mg /ml) using an increasing volume of 10-50 ul of stock solution . Hope this works for you.

Best regards

Hi Deepti,

thanx for your post, I'm using the same protocol ;), It use to work, but ...

Hi Hudec,

Thanks for your message... Then I cant find why this precipitation is happening...

...Hi Deepti, don't worry, me neither.

Fortunately by preparing filtrated 1x Bradford solution from the filtrated concentrate, the precipitates are less irritating and the slope in calibration approaches the correct values. Its not perfect, but acceptable ;)