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.

Working dilution for ELISA, IL and  IP needs to be determined experimentally.

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. 

collection of antibodies to various tags

Plant and algal protein extraction buffer

Secondary antibodies

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.