I am working on one protein which gets aggregated (similar to alpha-synuclein and tau) in certain neurodegenerative diseases. I am trying to confirm the aggregation using Western blotting. However, I am unable to capture aggregates in my blots. Can anyone suggest what kind of lysis buffer, transfer membrane, and transfer settings need to be used to see the aggregates in the stacking gel?
Thanks in advance.
Dear Kavikumar a K
I'm not sure that you can use WB to see the kind of aggregates that you would like to detect since to run WB you need to run a SDS-page with is denaturing approach that probably can destroind the non covalent aggregates. In case you have aggregation mediated by S-S bond as for example happen in ALS and sod1, you can try to run a non reducing SDS-page, followed by membrane transfer and detection by WB, but in case that aggregation is non covalent, i do not think you can detect it. Moreover also the trasfer of molecule at so high MW is very difficult. In case you have the recombinant protein and you would like to follow its in vitro aggregation you can monitor it by SEC or by fluorescence detection.
best
Manuele
Dear Manuele Martinelli
Thanks a lot for your answer. I suppose my aggregates are not covalently bound. I am going through the literature, and I found certain papers like this one - (Article Spatiotemporal analysis of soluble aggregates and autophagy ...
) 'where they run SDS gel followed by transfer, and they show that the aggregated proteins form a smear-like appearance in the stacking gel.
If I use non-reducing conditions, most of the proteins (including my housekeeping proteins) are stuck in stacking gel, so I am unsure whether this is the correct approach. Your views on this are welcome.
During our purification of proteins which are naturally prone to multimerization (like eg capsid proteins), we can observe these multimers in SDS-PAGE or rather Western Blot if we simply don't boil the samples prior to PAGE. Which by the way you don't have to do, if your analytical question permits it. The presence of increased salt concentrations may help, as well as excluding DTT.
But when you want to see whether multimers are formed, follow Kavikumars advice and do an analytical SEC with a concentrated sample, low flow rate and low injection volume via loop.
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