I am planning to do western blot to see the expression of my protein of interest in total cell lysate. Its always recommended not to freeze thaw a protein more than 2 times. But as a beginner I just want to know few things:
1. How many freeze thaw cycle a protein can tolerate and still can be used for western?
2. If freeze-thaw of a protein is done in ice whether still it will degrade after 2-3 cycle of freeze thaw?
3. Is there any storage condition in which a protein will be less prone to degradation after freeze thaw and can be used for western?
It depends on the properties of your protein. I have worked with proteins that (1) can be freeze thawed many times; (2) is aggregated if you freeze it; (3) remains soluble after thawing but shows not activity. So ...
If your sample is in SDS PAGE loading buffer already, don't bother about freeze thaw. It won't have any effect. But please check your reducing agent (just briefly smell its presence because both DTT and BME are really smelly), if it's gone then just reintroduce a bit of it.
Lesson learned from restriction enzymes: you can store them in high concentration of glycerol to store them in a freezer, they won't freeze. But be careful if your protein is sensitive to glycerol.
If your cell lysate is in something like Laemmlis sample buffer (containing sds) you can probably freeze thaw hundreds of times and still get a reproducible signal by WB. Just don't boil every time you thaw.
The number of freeze-thaw cycles a protein can tolerate depends on many things, like the protein itself, its purity, the solution it is in, and presence of protease inhibitors, etc etc. It also depends on what you want to do with it. If you're going to WB, then the fold of the protein is unimportant, but if you're going to use it for activity assays, then correct conformation is essential.
It depends on the properties of your protein. I have worked with proteins that (1) can be freeze thawed many times; (2) is aggregated if you freeze it; (3) remains soluble after thawing but shows not activity. So ...
If your sample is in SDS PAGE loading buffer already, don't bother about freeze thaw. It won't have any effect. But please check your reducing agent (just briefly smell its presence because both DTT and BME are really smelly), if it's gone then just reintroduce a bit of it.
Lesson learned from restriction enzymes: you can store them in high concentration of glycerol to store them in a freezer, they won't freeze. But be careful if your protein is sensitive to glycerol.
Addition of protease and phosphatase inhibitors to lysis buffer helps to reduce the protein degradation upon freeze thaw cycles. Every operation with proteins should be performed on ice (preferably in cold room). A practical advice to avoid freeze thaw cycles- prepare 25-50 ug aliquotes (as per your requirement) and store them in -20 or -80 freezer. Thaw the aliquotes you need and use the entire amount. In this way you will avoid freeze thaw cycles.
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