My question is about the storage of proteins (to probe for phosphorpoteins) after adding SDS buffer and B-meracptoethanol, reducing for 5 minutes at 95c, is the sample going to be stable at -20C? and do i have to boil at 95c before i load the protein? Thank you
Hi Aseel,
I don't see the harm of re-boiling protein samples at 95 degree C to ensure protein samples are completely denatured and ready to be loaded for reduced SDS-PAGE analysis (assuming you're doing SDS-PAGE reduced). It's a matter of 5 minutes!
Regards,
Harleen
Hi
usually If I'm not carrying SDS-PAGE on the same day after boiling for 5 min, I store samples at -20 and next day I re-boil for 2 min at 95 degrees C.
Hope this is helpful :)
Elham
Thanks everyone for answers, i ran the samples after i boiled them for around 3 minutes and it worked like a charm :) best of luck for all.
It is not essentially necessary to re-boil samples in this setting. However, it has no negative impact and may help to advance the outcome of WB after longer storange of samples in the context of complete denaturation and clear fractionation during SDS PAGE.
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