2 replies to this topic

[Kriz]

Hi! I have extracted protein lysate from tissues, did protein quantitation, added sample buffer and denatured the protein by boiling for 5 min and stored in aliquotes at -80 degrees. I reboil the sample again before loading. Will reboiling again before loading degrade the protein? I'm not getting any bands and staining with ponceau also reveals no bands though on quantification there was a gud amount of proteins?

[Curtis]

I usually save my samples in the same lysis buffer in -80. I keep aliquots but never add sample buffer to keep in freezer. Never had problem with proteins missing on membrane. How does your gel look like? do you stain it after blotting?

[nocas]

Kriz, on 31 August 2010 - 07:12 AM, said:

Hi! I have extracted protein lysate from tissues, did protein quantitation, added sample buffer and denatured the protein by boiling for 5 min and stored in aliquotes at -80 degrees. I reboil the sample again before loading. Will reboiling again before loading degrade the protein? I'm not getting any bands and staining with ponceau also reveals no bands though on quantification there was a gud amount of proteins?

Hi
I only denature the proteins once, either before the loading or in the day before (in this case I storage the sample at -80ºC after boiling until the next day). This works for me.

Edited by AnaG, 31 August 2010 - 07:59 AM.