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Protein stability - (Feb/28/2011 )

For my study, I proposed that our protein would have higher stability when treated with copper. So, I used Cyclohexamide to inhibit the protein synthesis. By doing this, I want to find a time point that this protein start to degrade and then I will add copper to see whether copper would increase this protein stability. But, the problem is once cell treated with the Cyclohexamide, most of the cells start to dye. When I harvest the survived cell and purify the protein which is resolved on SDS-PAGE. But there is no difference between the control and Cyclohexamide treatment group.

I want to know is there any method other than Cyclohexamide treatment could inhibit protein synthesis. Or is there other good method to test protein stability?
Thank you!

-liweixie-

YOu can do what is known as a "pulse-chase" where you pulse the cells with S35labelled Methionine after methionine starvation for 12 hours, then chase with unlabelled methionine again and look to see how long your protein exhibits radioactivity.

There are other drugs that will inhibit protein synthesis, but they are also toxic. Did you titrate the amount needed to ensure that synthesis was stopped?

-bob1-

I tried different concentration, but it seems that different concentration inhibit the protein synthesis would cause death. When I harvest survived cell to test the present protein, treatment has similar amount protein as the nontreatment.

Do you have any idea to test the protein half life except by using the radioactive labeled S. I am not permitted to work with the radioactive.

bob1 on Tue Mar 1 22:49:27 2011 said:


YOu can do what is known as a "pulse-chase" where you pulse the cells with S35labelled Methionine after methionine starvation for 12 hours, then chase with unlabelled methionine again and look to see how long your protein exhibits radioactivity.

There are other drugs that will inhibit protein synthesis, but they are also toxic. Did you titrate the amount needed to ensure that synthesis was stopped?

-liweixie-

Stable isotope labelling?

-bob1-

Yes


bob1 on Wed Mar 2 22:59:31 2011 said:


Stable isotope labelling?

-liweixie-