Hi there, I've been trying to detect oligomeric bands of alpha synuclein, but I'm not sure if it might work because I have been told that the size of aggregates (beta-amyloid or alpha synuclein in my case) is too large to even pass through any kind of gel !!
Any idea or suggestion is welcome. Thanks a lot!
You may be able to get something as large as a 4mer but above that the proteins would just sit at the top of the well. What exactly are you trying to determine?
I tried a similar thing 10 Years ago and it did not work. But well, if I started today I would consider the following:
- alpha Synuclein is a small Protein, so multimers (a-syn fibrills) should be able to enter into a low-concentrated gel. You could try a 4-6% Gel. But the huge a-syn aggregates will for sure not, as sarah mentioned, enter the gel.
- I suppose that you plan to do a western. If yes, be sure to use an antibody that can detect the native form of the Protein. (LB509 from Zymed works well in histology and detects aggregates, but it is a rather expensive compound, so i don't know if it is wise to use it for a blind shot).
- try a wet-blot procedure. Everything > 70kDa will blot suboptimal with a semidry setup.
Have Fun!
Imumunodetection of monomer and oligomer aSYN can be dramatically improved by membrane fixation with paraformaldehyde before probing.
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