probably DNA is disturbing the quantification of my sample
Posted 09 June 2010 - 07:42 AM
my problem is the following, i have to do 2D gel after immunoprecipitation of sample.
i have quantified my sample once, i made western blot on it with positive control, it seems that the amount loaded is not correct, since i couldnt identify band on my sample by WB, and the band is very visible in the control. even after transfert on membrane, i dont see proteins after rouge ponceau staining!
i tried to requantify, because i think that may be i have still DNA in my sample that mask the real absorbance of my sample by increasing it!
so, i decided today to add DNase to an aliqot, and keep one without dnase. when i quantified by bradford, i see difference, the sample with DNase is less concentrated!!
i dont know what to do know to my samples, if to treat them all with DNase, and requantify, then, perform IP and 2D gel??? this sample is very important, since it takes my big experience to obtain it, i dont want to take risk and lose it by performing directly IP with wrong concentration!
another thing, this sample containing membrane protein that i want to immunoprecipitate, was precipitated in solution with methanol, ethanol and acetone since 5 months in -20°C. are my sample degradated or something like that??
thanks for your interaction and advices
Posted 09 June 2010 - 05:24 PM
Posted 10 June 2010 - 12:51 AM
i added DNase to only one aliqot. i centrifuge for 15 min, and leave on ice for while, then, i requantify!
it seems that the concentration is the aliqot with dnase is low (2mg), and the aliqot without Dnase is 4mg!
i dont know how to reply to your question, but, im based on what i found!!!
unfortunantly, i'm not an expert on this
Posted 10 June 2010 - 01:58 PM
im sorry, you are right, i have inverted , it seems that sample with dnase is highly concentrated comparing to same sample without dnase.
but, i added 4ug of dnase in 60ul of sample from dnase stock 1mg/ml.
i requantified, i found again the double in sample with dnase,(same aliqot defrosted).
imay be its right, but, i think that i have DNA in my sample coming from brain tissue.
if u have any suggestion, thanks