Hello,
We are trying to detect the aSyn aggregates/oligomers /monomers in the AAV8-wt-aSyn mouse model. It is all about the technique details.
For the biochemical extraction part, I am considering whether use:
1. RIPA+1% Triton-100-->Triton soluble supernatant; Precipitation/insoluble-->10%SDS according to Sampson et al., 2016 cell 167,1469-1480. They used Thy1-aSyn mice.
Or
2. TBS-->soluble; insoluble-->TBS+5% SDS-->soluble; insoluble-->TBS+5% SDS +8M Urea according to Bandopadhyay R et al., J. Vis. Exp.
And for aSyn antibody choose, do you or someone in your lab have a recommended antibody for recognizing different forms/composition of higher order aSyn oligomers with a relatively good sensitivity in our model. Right now I have Syn211 (#AHB0261,Sigma) and 4B12 (#SIG-39730, Covance).
Thank you very much!
You are on a very interesting project. If you need to detect aggregates and oligomers of a-Syn, you want to make sure not to tease them apart by urea. I have personal experience in detecting oligomers in SDS-PAGE through WB (different proteins), and there is no golden rule since the dynamics of SDS-resistant oligomers differ from protein to protein. Just try different detergents such as NP40, TX100, deoxycholate and combinations. Compare DDT with B-Merc and without them before and after boiling, etc. Sometimes the addition of EDTA makes wonders.....
As for antibodies, there are 2686 a-syn antibodies across 55 suppliers, and narrowing it down delivers 857 mouse a-syn Western Blot antibodies across 38 suppliers. Aeonian Biotech is a new service to aid scientists finding what they need without running the risk spending a fortune in buying unfit antibodies.
Please refer Figure -6 [Not aSyn]
Details are in Materials and methods:Western blotting and densitometry section.
Please let me know if you have any questions.
https://www.researchgate.net/publication/292615481_Mitochondrial_oligomers_boost_glycolysis_in_cancer_stem_cells_to_facilitate_blebbishield-mediated_transformation_after_apoptosis
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