false results with the BCA assay? - (Aug/25/2005 )

hi

i have a problem with measuring the concentration in my lysates. i use the bca kit from Pierce and it shows that the protein concentration in my sample is very low or none. when i run the western, however, the protein concentration seems to be ok, the bands are strong.

it seems that something in the buffer disturbs the bca measurment. has anyone had this kind of problem? do you know what other method i could use? otherwise i can never load equal amount of the protein!!!
the method with running a gel with different dilution of the sample is not good for me, since i realy hav a lot of samples for each experiment

thanks for help!

Does this problem occur in all samples are just one? What is the composition of your lysis buffer? Are you using microplate protocol? I think that particular method is wildly inaccurate due to small volumes that the protocol calls for.

I use the standard protocol, run each sample in duplicate and at two different volumes.

hi Bosco!

my lysis buffer has 20mM tris (pH 7,5), 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton and proteinase inhibitors.

i use a 96 well plate. and the problem is with all the samples.
what is the standard protocol youre referring to?

cheers

I use the Pierce BCA assay. I had similar problems with the microplate so I switched to what they call the test tube procedure, or standard protocol. THe sample to working reagent is 1:20 instead of 1:8 in the micro assay. Basically I add 50 parts of Reagent A to one of B and this is the working reagent(WR). I add 60ul of my sample to 1.2 ml WR, incubate at 37C for 30min then read one tube at a time on a spec @ 562nm. Its slow and laborious, but the set up is easier than the microplate and where the microassay was notoriously inaccurate towards duplicating results, I've little error across different dilutions and/or duplicate samples.

I'm sure other people have used this micro-method to great success, but as they say go with what works for you. This happens to work for me.

Pierce has more info on the standard assay.

thannx a lot

i read pierce instructions and they say egta is interferring with the measurment, so that could be the reason, although when i used a different buffer before, wihout egta, i also had problems with the measurment