Can anyone help with a working protocol for amyloid beta detection in mouse brain by western blot? I've tried using two different antibodies (abcam and CellSignalling) but none of them worked. I was told that there may be a special way of processing the tissue in order to be able to detect amyloid beta in the hipocampus samples that I use. I have been using homogenates extracted with RIPA buffer and a protease inhibitor cocktail (Halt) from ThermoScientific.
Any help (including references) will be appreciated.
Hi Samuel
I had good results using 4G8 and 6E10 primary from Covance and goat antimouse IgG from Santacruz. But all the experiments I did were on cell lysates prepared using RIPA buffer. But the amyloid-antibody complex should be immunoprecipitated before loading onto gels. Probably that is where you are loosing protein. You can check for figure 3 in this paper Mol. Pharmaceutics, 2012, 9 (7), pp 1887–1897. Good luck.
Hi, for what I know you have to boil the membrane after the transfer so you expose the epitopes of the amyloid.
Hi Samuel, as Fernanda mentioned, boiling the membrane after the transfer in PBS for 5 min will surely help to expose the epitopes.
I've started a project on this site to share protocols and methodological details on amyloids. For now, it would be located on this site and be open to anyone who wants to join (don't put proprietary information in there that you do not want seen by others). Message me back if you want to join.
Hi Samuel, have you finally found a western blot protocol to observe ab1-42? I am interested too!!
Hi guys, do you need to build the membrane even if you pre-heat the samples with loading buffer at 60 °C as usual for WB?
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