If I boiled the protein samples for western blotting (After BCA and mixing with laemli and beta mercaptoethanol) at 95 degree for 5 minutes and store in -20 to continue western blotting later does it necessary to boil it again when need to do western later on as protein is already denatured and stored.
In other case if I boiled again before running the samples does it has negative effect?
Thank you in anticipation
Theoretically, boiling again shouldn't be necessary. However, it will ensure that all the SDS that may have precipitated in the cold will be dissolved. Don't boil for more than a couple of minutes in order to avoid SDS hydrolysis and acidification of the samples
Theoretically, boiling again shouldn't be necessary. However, it will ensure that all the SDS that may have precipitated in the cold will be dissolved. Don't boil for more than a couple of minutes in order to avoid SDS hydrolysis and acidification of the samples
Just gentle warming at 37C will be sufficient to resolubilise the SDS. No need to boil again. Over treatment of samples to boiling can cause problems as suggested and may even be a cause of aggregation
Thank you for your kind suggestion, I have another question after extracting cytoplasmic or nuclear protein extracts i.e. cellular fractions and following by BCA, what you suggest to dilute protein in distilled water or the respective lysis buffer? to get specific amount to run for western blotting. For example I need to use 25ug protein for each time western in 20ul volume and mixing with laemli buffer including b-mercapto still need to dilute so which option is best like cytoplasmic lysis buffer for cytoplasmic fractions or if I dilute in DW that's also fine.??
TIA
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