Hello, so, i just did a protein denaturation at 45ºC for 45 minutes in a thermal cycler for an electrophoresis for a posterior western blot. The problem is that i fu*** up the gels when i was about to load them, and now, because of the time, i'll have to do the electrophoresis tomorrow, but my proteins are already denaturated (and now are saved in a fridge at -80ºC). What should i do tomorrow with them? I'm thinking about denaturing them again because i think they might renature, but i'm not totally sure.
Thanks for any help and your time.
:(
I'd denature them again, to be on the safe side, as long as that does not interfere with the interpretation of your experiment.
If you have already denatured your proteins by incubating them at 45°C for 45 minutes and then stored them at -80°C, it's unlikely that they will renature upon thawing. The denaturation process typically involves the disruption of protein structures, and this is generally irreversible.
Here are a few suggestions for what you can do tomorrow:
Remember to maintain cold conditions throughout the process to prevent any potential degradation of your proteins. If you have concerns about the denaturation status, you can run a small test gel or perform a brief denaturation step before loading the full samples onto the gel. Always follow standard protocols for gel electrophoresis and western blotting to ensure reliable results.
If you have heated the sample for SDS-electrophoresis in traditional Laemmli buffer, you don't have to worry about renaturation of the sample because it is stored in the presence of SDS. Proteins will stay fully denatured.
If reducing agent like beta-ME or DTT was used, fresh reagent should be added and the sample heated briefly (80-90C for 2-3 min) after thawing to ensure that disulfide bonds in the denatured proteins are still fully reduced.
Why you did it at 45C for so long and not at the 80-97 C range a few minutes?? Yes , after freezing you have to heat it again before loading to avoid protein aggregation.
If your loading buffer has SDS it can precipitate at low temperatures and cause loss of protein visible on gel if only mixed and loaded onto the gel. I would re-heat the samples after thawing. People (myself included) store samples all the time at +4 °C or -20 °C. It's all good.
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