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dhawker

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Everything posted by dhawker

  1. I need to determine the Ki and IC50 values for a series of compounds with an enzyme I've purified. The problem is that I have to purify the enzyme from whole tissue (pig brain). The yield of the purification is really low, and the process is pretty laborious. I was wondering if anyone could suggest a method to determine these parameters that would use the least amount of enzyme possible. I have access to a 96-well plate reader, so I guess I'm think more along the lines of having to run the minimal number of + combinations as possible to obtain reliable data.
  2. You see, that's the problem...they were never isolated under denaturing conditions in the first place (at least intentionally). The purification protocol is extremely long and taxing and so if it is possible to renature I really want to give it a try. As far as I can tell the enzyme is intact, just inactive and denature (SDS-PAGE looks perfect). I know the yield may not be great, but it would be better than going through the entire protocol again. Supposedly it is possible to refold this exact enzyme, so assuming this publication is legitimate (which is still in question in my opinion since they won't return my emails) can anyone give suggestions???
  3. I don't really have biochemistry experience in this so I was hoping someone could give me suggestions. I have isolated an enzyme from whole animal tissue, but it lacks all activity. I ran a couple of native gels and it looks like it is denatured (bands appear the same as those boiled in sds buffer prior to loading). I have a paper where they refold the protein using urea, but they are refolding inclusions bodies from e. coli overexpression (long story why we use tissue...). First question, does refolding even work for proteins isolated from whole tissue? Almost all of the papers I have found are always working with proteins generated in ecoli. If it's still doable, I'll ask the rest of my questions... What is a good concentration to have my protein at before starting? Low? How low? In the protocol I have, they elute from their final column under denaturing conditions (8M urea), then dialyze away the urea step-wise (4, 2, 0 M). Would it be possible to just dialyze straight into 8 M urea, then follow their protocol? What would you recommend for dialysis protocol (sample volume v. buffer volume, dialysis duration, number of buffer exchanges per concentration, etc...) At this point I have a ton of pure protein... just denatured, so I have room to mess around with it I guess. Hopefully I'm not asking too much, but I would appreciate any and all suggestions. Thanks!
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