Dear all,
I am looking for a ELISA kit to measure the neuromuscular junction, so we decided CAF (agrin) was the one to use.
However, searching for a kit, everybody (at least 5-10 first pubmed references) seems to use the kit NO LONGER provided by Neurotone (Schieren, Switzerland).
Could anyone provide an alternative?
Many thanks!
Why not build your own ELISA assay? Just pick two antibodies that bind to different sections of your protein, from two different organisms (or simply use one that has an HRP directly on it). There are plenty of ELISA protocols in the literature that you could look at for suggestions on buffers and the like. It'd take some optimization, but would be far cheaper than a kit should you use it more than once or twice. And you could probably use the Neurotune assay manual for help in optimizing your own.
Dear William,
sounds a great idea but have no expertise in that proccess. Any reading I must do before other than the neurotune assay manual?
Many thanks!
Any primer on ELISA, even wikipedia, should be of help. Essentially, you're going to get a high-protein-binding 96 well plate, and coat the wells with an antibody for CAF (that's the plate the Neurotune kit provides, precoated). Then you'll block with BSA, then add your protein mix. Wash that out, and only CAF should be bound to the antibody. Add a second antibody for CAF that binds a different spot on the protein - it should ideally be from a different species, or have HRP on it already. If different species, then add a secondary, HRP tagged antibody. If HRP tagged, jump straight to developing with TMB or some other HRP reactive color agent.
Most of the trick is just optimizing how dilute the antibodies should be, the buffers you use, and how much protein to use. But again, many ELISA primers are around online, or any paper where a novel ELISA is used, can probably help with that.
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