bradford absorbance - (Nov/18/2010 )

hi all,
i did bradford assay to qantify my samples, i made a double for each concentration of the standard curve, and also for the samples.
what i see, is that the 2 absorbances of the same concentration are a bit different ! its strange, because i have the same volume in each cuvette!
there idnt good linearity in the stadard curve proportional to different concentrations!!
what could be the cause? my collegue think that this might be due to error when i prepare bsa curve, tips not changed, gilson....
what do u think?, everytime i have different absorbances read in different time!
i hope u could understand my message

Could be any one of those things you listed, but it is also quite likely that the differences you are seeing are a result of slight time differences between measuring the different samples - the reaction doesn't stop, so with increasing time, the samples get a bit darker.