Western blot using nanoCLAMP and anti-His-HRP

Abstract

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).

Instructions

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

Solution
PBS + 5% Milk
PBST + 5% Milk

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

Outcome	Title
Successful	Western blot analysis of recombinant NusA using nusA-A1(Cys) as primary and anti-His-HRP as secondary
Successful	Western blot analysis of recombinant SMT3 (yeast SUMO) using SMT3-A1(Cys) as primary and anti-His-HRP as secondary