His-tag | 6xHis (clone HIS,H8 / EH158)

KLH-conjugated synthetic peptide 6xHis

500 femtomoles of His-tagged proteins IsiA, NifH, PsbA, PsbB and PetC were loaded per gel well in Agrisera PEB extraction buffer.  Proteins were separated on  4-12 % NuPAGE PAGE Bis-Tris polycacrylamide gel (Invitrogen) and blotted 1h to PVDF. Blots were blocked with   for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat, anti-mouse IgG horse radish peroxidase conjugated, from Agrisera AS11 1772) diluted to 1:25 000 in 2 % ECL Advance blocking reagent for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was  5 seconds.

Apparent molecular weight of recombinant proteins: IsiA - 27 kda, NifH - 34 kDa, PsbA - 30-37 kDa, PsbB - 40 kDa, PetC - 23 kDa.

20 µl of media form Pichia pastoris culture overexpressed His-Tegged proteins  were separated on 12 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with  5% skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, from Bio-Rad) diluted to 1:15 000 in  for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL (GE Heltcare) according to the manufacturer’s instructions. Exposure time was 15 seconds.

Antibody is present in 10 mM PBS, pH 7.2

His-tag (6,8,10xHis) needs to be properly exposed to allow detection. To prevent target protein folding, extraction should be performed with 6 to 8 M urea or using TCA-acetone precipitation method. 

His-Tag is a polyhistidine tag which consists of 6 histidine residues introduced on N- or C-terminus of the protein. The polyhistidine-tag can be used for recombinant protein detection using specific antibodies and it is not conjugated to any dye or enzyme.