Can anyone help me with a working protocol for amyloid beta detection by western blot? I have extracted the whole proteins from the cells. For the Western blot I used a Precast gel 4-20% (in SDS condition) and then for the transfer I used the nitrocellulose membrane. For the immunoblotting I used anti-amyloid beta42, clone G2-11(Millipore) but it doesn't work. Anyone have some advices or protocol for me? Thank you!
I'm looking to develop such an assay for my work as well, so I'll follow this question. If the Tau is an aggregation-prone mutant (e.g. P301L, G272V, or K280-Deletion), it's possible that it may be resistant to solubilization in 2% SDS. If so, it may not even enter the gel, which would prevent detection. There are protocols for solubilizing such proteins via the inclusion of Formic acid (Link 1, though it's for PolyQ aggregates), and there are also oligomer-specific Tau Antibodies (Links 2, 3). Hopefully someone with more direct-experience comes along with advice.
Link 1: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633704/
Link 2: http://www.emdmillipore.com/US/en/product/Anti-Tau-T22-oligomeric-Antibody,MM_NF-ABN454
Link 3: https://www.abcam.com/amyloid-oligomers-antibody-ab126892.html
Do you mean that beta-actin or beta-tubulin doesn't even show a band? If so, until you get that well-behaved loading control working your Tau blot is meaningless. Failure of such a robust loading control is sign of s more-serious problem. If that's what's happening you might want to consult with a colleague who also does western to get some trouble-shooting help.
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