Hi,

I'm trying to quantify amyloid proteins from mouse brains. I've been using a protocol where frozen brains are homogenized in TPER buffer and ultracentrifuged at 100,000g for 1h to generate a soluble protein fraction. The pellet was resuspended and homogenized in 70% formic acid and ultracentrifuged with the same conditions above to generate an insoluble protein fraction. After FA centrifugation, the sample separated into a floating layer (which looks like a lipid layer) on top of a clear FA portion and a pellet. The protocol says to use the supernatant as the insoluble protein fraction and there is no mention of this floating layer. I'm unsure whether to mix the floating layer and the clear FA portion and use that as my sample or whether this will affect the readout? I am going to be quantifying using an MSD plate for total Abeta. I have tried samples where I have directly centrifuged after homogenizing with FA and some where I have placed samples on a shaker for 1h before centrifugation and the same separation of layers has happened both times. I would appreciate any input on this problem.

Thanks!