Supersignal west femto maximum sensitivity substrate enhanced chemiluminescent substrate

Nitrocellulose membrane was marked with pencil to indicate the regions where the peptides would be spotted. Four micrograms of recombinant peptide was spotted onto each marked region of the nitrocellulose membrane, then the membrane was cut into strips and blocked in Tris Buffered Saline with Tween 20 (Cell Signaling Technology) and 5% bovine serum albumin (TBST 5% BSA) for 30 min at room temperature. The membrane was next incubated in cell culture supernatant from recombinant antibody expression diluted 1:20 in TBST 5% BSA for 1 hour at room temperature. Nitrocellulose was washed three times with TBST for 5 min per wash, then incubated with anti-human IgG-HRP (Southern Biotech) diluted 1:500 in TBST 5% BSA for 1 hour at room temperature. Nitrocellulose was washed three times with TBST for 5min per wash, then once with TBS for 5 min. Nitrocellulose was incubated with SuperSignal West Femto Maximum Sensitivity Substrate enhanced chemiluminescent substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions and chemiluminescent signals were captured using a CCD camera-based imager.