3 replies to this topic

[kartiga natarajan]

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?

[mdfenko]

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).

[kartiga natarajan]

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.....

[mdfenko]

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)