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Concentrating a diluted protein and protein determination

protein concentration precipitation

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4 replies to this topic

#1 madsmad

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Posted 10 July 2012 - 12:24 PM

Hi,

After using Bradford method of protein determination, I diluted the samples with water to bring a final concentration of the samples to 1 μg/μl. However, after doing a gel electrophoresis for control with Beta-Actin, I came to know that my samples have different amount of proteins, probably due to an error in the bradford method.
I want to determine the protein concentration again in my diluted samples. Is that possible even after adding sample buffer? If yes, how?
If not, how do I concentrate the proteins in my sample and re-determine the protein concentration?

Thanks

#2 doxorubicin

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Posted 10 July 2012 - 01:47 PM

Sample buffer is not compatible with Bradford. You might be able to use a complicated protocol (i.e. TCA precipitation) to get the proteins back into a Bradford compatible buffer, but it would be too much trouble. Why not just repeat the experiment and make new samples? Also, rather than diluting in water to equalize the concentrations, you should dilute the samples in lysis buffer, so that all samples will have precisely the same concentration of protein and salts etc. If your samples are very precious and you absolutely have to salvage them, your best bet would be to scan the actin band and quantify it using ImageJ to estimate the relative protein concentrations.

#3 madsmad

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Posted 10 July 2012 - 09:36 PM

Thanks doxorubicin. How about LOWRY or BCA protein assay? Is sample buffer compatible with these methods?

Edited by madsmad, 10 July 2012 - 09:56 PM.


#4 doxorubicin

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Posted 10 July 2012 - 11:19 PM

SDS seems to cause trouble for most of these assays, but I am not an expert. This modified Lowry seems like a possibility: http://www.cmdr.ubc....ROTEINASSAY.htm

#5 madsmad

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Posted 13 July 2012 - 10:33 AM

I'll try that and leave feedback soon. Thanks





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