Cell lysate at RT - (Apr/12/2008 )
Hi - I accidentally left my cell lysate at room temperature in the hood for two days; the lysate can be stored in 4C for one month or so and the proteins will be fine. But do you the lysate I left in the hood still okay, to run a Western?
I think that if you have added sample buffer and boiled it for 3 minutes @~100C then you have some chances.If i were you i would have done a new one without a second thought.Someone correct me if i am wrong,but i think that at least you will not detect all the phosphorylated proteins that existed originally in your sample.If you haven't added sample buffer,just throw it away.
If You are Purifying an Enzyme , Its better to discard the sample and start with a fresh sample. Always maintain good chilling conditions when purifying protiens. Proteins will undergo degradation at room temperature also(Depends on the protein which you are trying but mild degradation will be there and more over the problem with proteases)
To detect your sample degradation Run a westrn blot and see if you are getting good back ground then throw the sample, if not use the sample for next one step and run again a westrn to confirm the same .
Correct me if iam wrong
I used the "bad" sample with some other samples, but nothing showed up for some reason, so I dunno what went wrong. Oh, well, at least I know to remember to keep these things in 4C next time.
proteolytic degradation will be the most relevant problem; I would throw the samples away, and take a fresh preparation