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measurement of proteins in a mixture of soluble-insoluble proteins


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#1 marsiles

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Posted 18 September 2012 - 04:32 AM

Hi everybody.
We have obtained a purified his-tag protein after solubilization of inclusion bodies with 8M urea. We eliminated the urea and left between 1-3% triton x-100. Now, we are measuring protein content by BCA, using the same solution where we have the purified protein to prepare BSA curve samples.

BCA says I have let's say 1 microg/microl. When we load the same sample in an acrylamide gel (denaturing+sds) and stain with coomasie blue, also loading 0.5, 1 and 2 bsa microgram samples in the same gel, cisual comparison says that our protein is much less concentrated than the BCA says (around 5 to 10 times less).
As far as I can see, samples in commasie-stained acrylamide gels, after boiling and use of mercapto plus SDS could show HIGHER concentration in a coomasie gel than the same sample in a BCA that measures only the protein in solution....Or not??

I have gone through protocols and calculations, but I cannot understand were the failure is.

I need to know the concentration of the soluble protein, we need to immunize animals with it.

If you could help me, I'd be grateful....

#2 mdfenko

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Posted 18 September 2012 - 06:51 AM

besides the fact that bca is affected by triton (and you appear to properly take care of that with your standards),

coomassie binds differently to different proteins. bsa binds more coomassie than most proteins.

do you use g250 or r250? r250 is less affected by different proteins.
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#3 marsiles

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Posted 18 September 2012 - 07:13 AM

Yes, I read that triton affects BCA above 5%, I tried to stay below that, but still using it in the standars.
Coomasie is R. But differences in binding between BCA and Coomasie can be so accentuated??
Anyway, if I accurately want to know how much of soluble protein I have there....what should I believe? Coomasie? BCA? We also have a nanodrop, but I don't have a good opinion about nanodrop....

#4 mdfenko

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Posted 18 September 2012 - 08:12 AM

as long as the readings are within the linear range and are properly blanked the bca is probably more accurate.

and, you can check with the nanodrop (use 2ul, 1ul does not give consistent readings).
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#5 marsiles

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Posted 18 September 2012 - 08:38 AM

thanks. I'll try nanodrop with 2 ul and see.
I'm also concerned with the stuff of differentiate between soluble and insoluble proteins. As far as I understood, BCA only binds to proteins in solution, but not to proteins in suspension? What about the nanodrop, it only measures soluble proteins?

#6 mdfenko

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Posted 19 September 2012 - 07:30 AM

bca, like lowry and biuret, is a destructive method. the naoh in the medium breaks peptide bonds. any protein in suspension should be properly accounted for in the result.

coomassie and uv methods are non-destructive and are more accurate when used for solubilized proteins.
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