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

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AS11 1771
  | Clonality: monoclonal  |  Host: Mouse  |  Reactivity:His-tag

His-tag | 6xHis  (clone HIS.H8 / EH158) in the group Antibodies for Human/Animal  / Tag Antibodies / His at Agrisera AB (Antibodies for research) (AS11 1771)

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How to cite this product:
Product name, number (Agrisera, Sweden)

Data sheet Product citations Protocols Add review

Product Information

Immunogen

KLH-conjugated synthetic peptide 6xHis

Host Mouse
Clonality Monoclonal
Subclass/isotype IgG2b
Purity Protein A purified
Format Liquid
Quantity 50 µg
Reconstitution For reconstitution add 50 µl of sterile water.
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. For storage at 4°C for several days to weeks, sodium azide to final 0.1 % can be added. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Dot Blot (Dot), ELISA (ELISA), Immunoprecipitation (IP), Immunlocalization (IL), Western blot (WB)
Recommended dilution 1 : 1000 (WB)

Reactivity

Confirmed reactivity 6xHis
Predicted reactivity 6xHis
Not reactive in No confirmed exceptions from predicted reactivity are currently known.

Application examples

Application examples

application example

western blot on plant recombinant proteins using anti-His antibody

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.

 

western blot using anti-His antibody from Agrisera


20 µl of media form Pichia Pastoris culter 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.

M- protein ladder (Fermentas)
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

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

Related products

Background

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

Selected references López-Vidal et al. (2020). Is Autophagy Involved in Pepper Fruit Ripening? Cells, 9 (1) , DOI: 10.3390/cells9010106
Tan et al. (2020). Salicylic Acid Targets Protein Phosphatase 2A to Attenuate Growth in Plants. Curr Biol. 2020 Feb 3;30(3):381-395.e8. doi: 10.1016/j.cub.2019.11.058.
Häggmark-Månberg et al. (2016). Autoantibody targets in vaccine-associated narcolepsy. Autoimmunity. 2016 Sep;49(6):421-433. Epub 2016 May 20.

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