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protein extraction and DNP

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#1 kingah



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Posted 22 June 2009 - 03:36 AM

I have been having lots of problems with my samples and reaction I need to test recently so I decided to ask for some help from more experienced people :o
In general the aim of experiment is to check the carbonyl groups content in proteins from photoreceptors outer segments. I just isolate outer segments and have them in PBS solution. Then to extract protein I used Folch's method and got an interphase with proteins. Problems start at this stage :( . I need to dissolve my protein to perform reaction with 2,4-dinitrophenylhydrazone (DNP, it is in acidic solution with ethanol) and then get rid of excess of DNP and re-dissolve my proteins in order to measure spectra. I can also perform reaction with DNP with the pellet I get and then centrifuge my sample, followed by dissolution of proteins. I have tried to dissolve protein in 6M guanidine. I have also tried to use trypsin to dissolve it. Nothing worked.
Could anyone suggest some solutions? I am happy to try different methods.
I would be very grateful for your help.

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