Hi all,
I am attempting to extract AB-42 from some of my 10 month old J20 mice for an Abeta ELISA. I initially perfuse the mouse with heparinzed PBS then snap-freeze one hemisphere on dry ice. The hemisphere is then homogenized using TPER buffer and the homogenate spun down at 16,000g for 15 min at 4 C and the supernatant stored at -80 C. The remaining pellet is then homogenized in 5 M guanidine-HCl buffer and then kept on a nutator overnight at 4 C to complete homogenization. The homogenate is spun down and the supernatant is kept at -80 C.
I perform ELISAs using the Human AB-42 Elisa kit from Thermo using 1:5-1:100 dilutions of my homogenate in standard diluent buffer (kit component). Standards are run using same buffer as samples. My standard curve looks fine but I get no detectable signal for my samples. The two step extraction that I follow is fairly common in the field and the mice should have pronounced pathology by this age.
Any thoughts would be appreciated. Would concentrating the protein help?
Please try again, perform ELISA with A-beta superntant without dilution.
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