Dealing with Lavage Samples - (Aug/11/2010 )

Hi everyone,

New to the forum.

The post-doc under whom I work got quite angry at me today. He has a bunch of mouse bronchoalveolar lavage samples, diluted (I think) in PBS, which we normally keep at -80C but which I removed from the freezer and placed at room temperature in order to thaw them to perform an ELISA... About two hours after I thawed the samples, he came in and noticed that I had not placed the samples on ice and yelled at me saying that this could possibly damage the proteins in the sample. We are assaying for mouse CXCL5, which the other post-doc in my lab tells me is very heat labile.
They claim that these samples are irreplaceable, and I'm afraid that I may have messed everything up. I have only been working here for about a month, too.

Question is this: Is there any truth to the claims of protein degradation?

Please let me know.

- ice

Yes, protein samples will degrade quite rapidly at room temperature. Most of the degradation is from proteases present in the sample and the rest due to freeze/thaw cycles. To inhibit some of the degradation, thaw on ice and add protease inhibitors.