To demonstrate the specificity of the phosphorylation site specific antibody ( ab4803 ) a 1/150 dilution of anti human-FAK (phospho Y397) antibody was pre-incubated with 200 fold molar excess of peptide (ab40145, 1/15 dilution) for 12 hours rolling at 4°C. Human Tissue sections (kidney) were deparaffinized, rehydrated and antigen retrieval was performed using citrate buffer pH 6.0. Endogenous peroxidase was quenched using 3% hydrogenperoxide in methanol and blocking was performed. Primary antibody alone (positive control) or mixed with peptide (blocking control) or antibody diluent alone (negative control) was applied to tissue sections and incubated for 16 hours at 4°C. Detection was performed using a HRP detection system. Nuclei were counterstained using Hematoxylin.
Performed under reducing conditions.
Performed under reducing conditions.