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Quantitation of protein concentration in rehydration buffer

membrane proteins

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3 replies to this topic

#1 kartiga natarajan

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Posted 15 April 2013 - 03:40 AM

I isolated membrane proteins from Thermo Scientific kit...Due to detergents in the reagents I did methanol chloroform precipitation after the isolation and since i have to do 2D...I resuspended the pelleted in rehydration buffer without ampholytes & DTT...

To determine its concentration,what shall i can do without purchasing the 2D quantikit? Is there any other buffer i can dissolve the pellet to quantify its concentration before doing 2D?

Please help me out regarding this?

#2 mdfenko

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Posted 15 April 2013 - 04:06 AM

not knowing the composition of the rehydration buffer, it is hard to determine which protein assay to use.

but, since there are neither ampholytes nor dtt present, bradford, bca, or lowry should work (with proper blanks).

absorbance at 280nm may also be okay if you only require a rough estimate (again, with proper blanking).
talent does what it can
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#3 kartiga natarajan

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Posted 15 April 2013 - 10:31 PM

Thanks for your reply....The composition of my rehydration buffer which i have used as a sample buffer contains 7M urea, CHAPS and bromophenol blue....Will bromophenol blue interferes with bradford assay? Also, in bradford assay what blank i should use? Bradford alone or along with my rehydration buffer...If so also tell me the dilution.....

#4 mdfenko

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Posted 16 April 2013 - 03:37 AM

the bromphenol blue pretty much rules out using colorimetric methods.

absorbance at 280nm may work with proper blanking but i wouldn't count on it.

you might be able to use a fluorescent protein assay (using fluorescamine) or an infrared method (requiring the purchase of this instrument from millipore)
talent does what it can
genius does what it must
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