storage of protein samples - (Apr/19/2009 )

I was just wondering if anyone knew of any problems storing protein samples that have been boiled (5 mins) in SDS sample buffer at -20 degree. I have some samples that I ran which would have been absolutely perfect if my loading hadn't been slightly off. I had stored the samples at -20 for just a few days. Took them out, thawed and rewarmed for 1 minute in a heat block. Spun down for a few minutes and reloaded a gel with the correct loading. Now, although my loading is dead-on, the resulting westerns are giving me completely different results?!? Seriously, how can the same samples give me night and day results? By the way, I'm using the same antibody dilutions for both westerns.

I've never had any problems like that. I would have re-boiled the samples though, just to make sure that they were denatured.