Procedure For Analyzing Tissue Homogenates In The Competitive EIA

Preparation of Tissue HomogenatesFreeze tissue @-70OC for 2-18 hours Suspend tissue in 0.5-1.0ml of Tris Buffered Saline (TBS), pH7.4 and quickly sonicate the preparation. Centrifuge at 5000 rpms for 15min Take 50µl of supernatant and dilute it 1:20 or 1:50 in TBS and set aside in ice bath. This aliquot will be used for protein concentration by determining OD@280nm. Dilute the remainder of the supernatant 1:2 in kit Diluent #2 Perform 2-fold serial dilution if necessary. Additional serial dilutions should be made with kit Diluent #2

Assay ProcedureAdd 100µl of standard or sample to designated wells. Reconstitute antibody and conjugate per the kit instructions and follow normal assay procedure. Following this protocol, your resulting concentrations will need to be multiplied by a dilution factor of 2. Normalize potency estimates for protein concentration