Western blot using nanoCLAMP and anti-His-HRP


This protocol describes the blotting and detection of proteins bound to PVDF membranes using nanoCLAMP(Cys) as primary antibody and anti-His-HRP (Sigma A7058 or equivalent) as secondary, developing with TMB for membranes (Thermo).


1 Prepare lysate or solution containing target protein in SDS-Sample buffer and separate by SDS-PAGE
2 Transfer protein to PVDF membrane by semi-dry transfer or tank transfer, following transfer apparatus' instructions
3 Rinse blot with ddH2O to remove any gel debris, and allow blot to dry
4 Wet the blot with methanol, then submerge in ddH2O
5 Block the blot in 5% milk-PBS on orbital shaker for 1 hour at Room Temperature
6 Pour off the blocking solution, and add nanoCLAMP(Cys) diluted to working concentration (approximately 0.25 ug/ml) in 5% milk-PBS-T (0.1%) on orbital shaker for 1 hour
7 Pour off the primary solution and wash the blot with PBS-T(0.1%) for 5 seconds. Repeat this step for a total of 3 times
quick rinses to remove milk
8 Wash the blot again with PBS-T(0.1%) for 5 minutes. Repeat this step for a total of 3 times
9 Remove blocking solution, and add anti-His-HRP (Sigma A7058 or equivalent) diluted to 1:4000 in 5% M-PBS-T(0.1%) on orbital shaker for 1 hour
10 Wash blot as described following primary incubation
11 Add TMB for membranes (Sigma) to develop

Solution(s) Used

Outside Material(s) Used
Vendor Outside Material Catalog Number
Sigma-Aldrich anti-His-HRP A7058
Sigma-Aldrich TMB for membranes T0565-100ML
Applications Using This Protocol