ELISA vs Western - The two aren't agreeing.. (Apr/08/2008 )
I've been running a bunch of Westerns lately to examine APP processing and levels of abeta in mouse brain tissue. I am now supposed to run Abeta[1-40] ELISAs (using the kits from Biosource/Invitrogen) on the same mouse brain tissue to get a better idea of the levels of abeta.
However, when doing the ELISAs, I get absolutely no signal from any samples.
Initially, I tried using the raw extract, which yielded nothing significant. Ever since, I've been using the guanidine purification technique, with a final concentration of 0.05 guanidine by the time it reaches the plate (I've tried a couple different protocols* and concentrations; recommended concentration is 0.1M or less), but still I get nothing.
I'm not sure if I should try something else or if I'm doing something wrong, but any insight would be much-appreciated.
*Protocols, for those interested --
1. Add equal volumes of cold 5M guanidine and previously-extracted brain homogenate to a new tube. Incubate at room temperature for 1h. Spin down at 20,000xg for 30m. Take off the supernatant and dilute 1:50 with supplied ELISA diluent buffer. Proceed with ELISA protocol.
2. Add 8 volumes of cold 5M guanidine to brain protein pellet and homogenize with a Dounce homogonizer. Transfer to tube and incubate while shaking for 3.5h. Spin down at 16,000xg for 30m. Take off the supernatant and either freeze or dilute 1:10 with 5%BSA/0.3%Tween/PBS. Dilute further 1:5 with supplied ELISA diluent buffer and proceed with ELISA protocol.
do u have any positive control in ur ELISA? if yes make sure it is working properly. if it is working properly then u can say that your isolation proces is effecting the binding of amyloid beta peptide to the antibody.
i would also suggest that, use pure or abundant APP protein for ELISA. denature the protein by boiling with mild detergent and DTT. load it in to ELISA. this will confirm that you isolation process is effecting the ELISA.