protein stability issues - (Jun/25/2007 )

For the last couple of months, I have been doing filter binding assays with hsp90, a chaperone protein. But recently for the last couple of weeks, my experiments have no longer been working... I've been trying to think what I suddenly changed in my protocol, and one thing that popped in my mind was vortexing my protein solution.

Does vortexing (on our vortex it is quite vigorous) denature proteins that much? Should I just invert a few times to mix in the future?

Any comments would be extremely helpful, thank you!

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vigorous shaking may be harmful for some proteins, especially larger protein complexes; it depends on duration and how strong you vortex; there is another point: by vortexing you increase contact of proteins with oxygen which could oxidize functional groups;

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Our vortexer is quite strong, so when I vortex a solution, essentially a column of air forms in the middle of the tube. My protein is about 25 kDa, and I have always had difficulties with its stability. Before I used native purification processes, I used to do denaturing purification, but my protein just would not fold at all even with 7 day dialysis with Roche dialysis cassettes.

Thanks for your comments The Bearer, I will make new protein and try only inverting to mix...hopefully that will solve the problem...

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So, do you remember how exactly you treated your extract before you start vortexing? It would be better to keep a standard protocol ,and it is good that you point this out. You could also use a plastic homogenizer for eppendorfs to slightly lyse your extract before an 15-30min incub on ice and then centrifuge, but I dont know your exact protocol.
Hope I was of some help!

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Hi Seraph524!

Vortexing protein is not a solution of problems with solubility of your protein. This procedure is not used in protein work because during this process denaturation of protein will occur. I think you should improve your refolding step procedure to increase solubility and stability of your protein?

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