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SDS-PAGE w/ PFA fixed tissue


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#1 LacquerHead

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Posted 21 June 2012 - 12:29 PM

Hi all,

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?

Thanks!

#2 Maxter

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Posted 21 June 2012 - 01:13 PM

The way I had it understood is that once you fix with PFA, there is no way back, as you are forming a series of covalent bonds that link peptidyl amino to peptidyl amino goups (I don't think BME would reverse it as it does with disulfide bonds). Boiling does not provide sufficient energy to break covalent bonds (but it does break non-covalent interactions). For the SDS-PAGE to work, remember we add the SDS to effectively destroy the 2º, 3º, 4º stuctures (in other words, the protein has to be a string with SDS attached around it, giving it an overall negative charge). Because you have it crosslinked, the protein has its higher order structures intact. It seems like the stacking gel lattice is not small enough to hold your protein from moving, but the resolving gel has too small of a lattice (12% concentration) for the protein to go through in the folded form. If your antibody has specificity for the folded form of the protein, I probably would make a big stacking gel (I don't know what concentration you are using) and run it, then transfer it to the membrane (I would not include a resolving gel). This would be my quick-and-dirty approach...

#3 LacquerHead

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Posted 21 June 2012 - 04:41 PM

The way I had it understood is that once you fix with PFA, there is no way back, as you are forming a series of covalent bonds that link peptidyl amino to peptidyl amino goups (I don't think BME would reverse it as it does with disulfide bonds). Boiling does not provide sufficient energy to break covalent bonds (but it does break non-covalent interactions). For the SDS-PAGE to work, remember we add the SDS to effectively destroy the 2º, 3º, 4º stuctures (in other words, the protein has to be a string with SDS attached around it, giving it an overall negative charge). Because you have it crosslinked, the protein has its higher order structures intact. It seems like the stacking gel lattice is not small enough to hold your protein from moving, but the resolving gel has too small of a lattice (12% concentration) for the protein to go through in the folded form. If your antibody has specificity for the folded form of the protein, I probably would make a big stacking gel (I don't know what concentration you are using) and run it, then transfer it to the membrane (I would not include a resolving gel). This would be my quick-and-dirty approach...


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.

#4 Maxter

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Posted 21 June 2012 - 08:20 PM

Did you try labeling with the antibodies anyways? It might work... (you won't know until you try)
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!

#5 mdfenko

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Posted 22 June 2012 - 08:48 AM

i don't know if they only work for immunohistochemistry or if you will be able to obtain sds runnable samples but there are methods for antigen retrieval from formalin fixed tissues.

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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