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losing protein activity


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#1 BURCU:)

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Posted 04 November 2004 - 02:04 AM

Hi all! I have a problem in protein purification.I used size exclusion chromotography to purify my protein but after purification I lose the activity of my protein.It isn't because of proteases or other inhibitors I know that but what can be the other causes?what should I do? thank you :)have a nice day...

#2 Janina

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Posted 04 November 2004 - 04:36 AM

What about degradation of your protein by your bacteria? Perhaps they don't like it... Or check the protein folding resulting of the sequence (degenerate codons?)...

#3 Sprag

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Posted 04 November 2004 - 08:24 AM

you could be loosing some co-factors, or ATP/GTP, or NADPH..
or simply denaturing of your protein, or change in pH...

that's for you to find out...

soren

#4 BURCU:)

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Posted 04 November 2004 - 11:01 AM

What about degradation of your protein by your bacteria? Perhaps they don't like it... Or check the protein folding resulting of the sequence (degenerate codons?)...

I try my protein on sensitive strain S. cerevisia so it's sensitive to my protein.protein kills S. cerevisia before chromotography therefore protein lose its activity in column.it's active in pH 4.5 so there is no much chance in means of buffer.pH is limiting me about choosing buffer.I use sodium-phosphate buffer.And another problem is that;in chrotomography the peaks are very close each other I can't seperate them easily There will be always a contamination from other protein.please help me. I hope I could explain my problem clearly:)




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