SDS-PAGE w/ PFA fixed tissue
Posted 21 June 2012 - 12:29 PM
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 Im 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?
Posted 21 June 2012 - 01:13 PM
Posted 21 June 2012 - 04:41 PM
Thanks for the advice, unfortunately my antibodies all recognize linearized epitopes so that approach may not work. Going to have to to think of some other way to break the specific covalent bonds created by the addition of PFA.
Posted 21 June 2012 - 08:20 PM
In terms of breaking the linkages, check this bond energies page: http://www.cem.msu.e...ge/bndenrgy.htm
it seems that, at least in terms of energy (adding heat = boiling under high pressure), you won't be able to break the PFA crosslinking without breaking down the peptide bonds in your protein... I don't know if there might be an enzyme that likes digesting this kind of ester bonds (some kind of esterase?). The crosslinking seems to be O-C-O type... good luck!
Posted 22 June 2012 - 08:48 AM
one of them is here: journal of histochemistry and cytochemistry (full text).
another (newer) method is here: methods in molecular biology (abstract).
here is another from jhc: reversing the effects of formalin fixation...
as a general caution (not regarding formalin fixed), prolonged heating of sds samples (in sample buffer) can lead to aggregation of the proteins. if necessary to heat for more than 3-5 minutes, it is recommended that the sample be incubated at 60-70C for 10-20 minutes.
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