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How to quantify the protein amount for serum samples ?


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#1 itaimo

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Posted 12 December 2011 - 04:00 AM

I have to do a western blot test to serum samples.
How can i quantify the amount of the total proteins to ensure equal loading ?

I'm not sure if a Bradford protein quantification test (BCA kit) will work well because that there is a color difference between the samples that could effect the test results.


Blood plasma samples were produced from blood samples using 0.5% Heparin solution and centrifugation.

#2 mdfenko

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Posted 12 December 2011 - 08:26 AM

first, bradford and bca are not the same.

since you have interfering color you will not be able to use methods in which you determine absorbance. you may be able to use the biuret method but it is (a lot) less sensitive than the two you mentioned (which may be okay for serum and plasma) but may also be affected by the intrinsic color of your samples.

you may be able to use a method that uses fluorescence, it may not be quenched by the interfering color.

one fluorescence method for protein determination uses fluorescamine. it is very sensitive so you will have to use very small amounts of serum (or plasma? you mention both in your post).

here is a link to a comparison table of protein assays (absorbance and fluorescence based):

invitrogen/molecular probes table 9.1
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