I am trying to run an SDS-PAGE gel 12% for p53 and alpha-actin and running into some trouble. I am using tissue samples that were fixed with PFA and this is likely the cause of the issues I'm having. BCA assay does not work, likely because of protein cross-linking due to PFA so I have to do a western for actin to get protein concentration, however even after boiling the samples and using SDS as well as BME I get no bands at all or weak bands.
In many membranes I see protein aggregates at the transition from the stacking to resolving gel and think this is a migration issue due to PFA cross-linking and creating protein aggregates. I am boiling my samples for 10 minutes, thinking of increasing this or doing a commassie stain to look for abnormal migration.
What does everyone think? Any experience with this? Suggestions?
If you are getting weak bands, then there is a probability that the gel is fine.make sure that your sample is properly lysed. In case of tissue samples the lysis is different from that of cell lines. Make sure you are using properly lysed samples. For p53 you can use 10% gel. Use a protein ladder in the gel. so that you can verify whether there is any problem with the gel or not by observing its migration!!!!
No ways to fins a solution: fixation is and irreversible process by generating irreversible crosslinks between proteins (covalents) and digestion with proteases cannot help, unfortunately...
Have you thought about reversible crosslinkers, such as those used in chromatin immunoprecipitation? I don't think you can resolve PFA fixed proteins.
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