His-tag | 6xHis (clone HIS.H8 / EH158)
AS11 1771 | Clonality: monoclonal | Host: Mouse | Reactivity:His-tag

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Product Information
KLH-conjugated synthetic peptide 6xHis
Reactivity
Application examples

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.
2- 20ul of medium before induction (PcGCE protein)
3-20ul of medium before induction (PcGCE S217N protein)
4-20ul of medium after 48h of induction (PcGCE protein)
5-20ul of medium after 48h of induction (PcGCE S217N protein)
6-20ul of medium after 96h of induction (PcGCE protein)
7-20ul of medium after 96h of induction (PcGCE S217N protein)
Courtesy of Dr. Marta Derba-Maceluch, UPSC, Umeå
Additional information
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.
Background
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.
Product citations
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