Hello,
I am trying to treat protein lysates before performing copper-based click chemistry to reduce non-specific incorporation of my alkyne into my azide-labeled proteins. Right now I am using lysate obtained in RIPA buffer. I am diluting the lysate in PBS, then treating with DTT 10 mM for 15 min at 80C. I allow the proteins to cool at room temperature for 15 min, then add iodoacetamide for a final concentration of 40 mM for 30 min in the dark at room temperature.
After this step, I see a lot of precipitate in my tubes and am wondering why this is happening/if it is avoidable.
Any help you could provide would be great!
Hello Emily,
When does precipitation occur ? My guess would be that heating the lysate at 80 °C would be sufficient to denature and precipitate most proteins, unless you are working with a hyperthermophilic organism. Denatured, precipitated proteins proteins can be brought into solution in the presence of strong chaotropic agents like > 6 M urea or 5 M guanidine hydrochloride, or 1 % sodium dodecyl sulfate, but I don't know whether this would be compatible with the next steps of your experiments.
IAA is an alkylation reagent,
the proteins are more hydrophobic after treatment with it
You should maybe consider to use less concentration of IAA
Hi Emily Norton . You are probably getting precipitation of DTT (Dithiothreitol) reacting with iodoacetamide. Switching to TCEP instead of DTT might solve the issue.
Did you resolve the problem? if so... how? it might be that your sample is too concentrated in protein. try diluting it at least ten times and see what happen..
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