Methods for detecting aggregated alpha-synuclein species in mammalian cells?

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Mohamed Khashan

Hi Naomi,

Pls refer to the following:

https://star-protocols.cell.com/protocols/509.

Samir Ranjan Panda

Hi Naomi Thorne

Please find the attached protocols.

1. Article Detection of alpha-synuclein conformational variants from ga...

2. Article Detection of pathological alpha-synuclein aggregates in huma...

Thanks

Mathangi Palanivel

I am trying to do this too and was thinking of employing Immunofluorescence to do this. My proteins are tagged to EGFP, so I would co stain the cells with both anti-EGFP and anti-pSer129 antibodies to observe if there is an increase in the aggregated version of protein simply by over expression. Do you think this might be feasible for you? Naomi Thorne

Naomi Thorne

Mathangi Palanivel thanks for your answer. I too have EGFP-tagged alpha-synuclein, and have done co-staining for GFP and pSer129 with immunofluorescence. I definitely see an increase in the staining, but don't feel confident in saying that what I see is definitely aggregation, since species expressing pSer129 are not necessarily aggregated. Though this information is useful, I am still looking for a definitive way to prove the presence of aggregates! I know that some antibodies exist that recognise only aggregated alpha-synuclein species, but I don't know how reliable these actually are.

Naomi Thorne Oh I see! Sure, if you do find a more definitive way, please let us know too:) I’m also trying out western blotting with both SDS- and native-page as affirmative experiments in addition to the staining.

Also I think the aggregates specific antibodies are worth a try, if you have the means to do it, do try it out:) You might have come across this publication on it : Article Conformation-specific Antibodies Targeting Aggregated Forms ...