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His-tag | 6xHis

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AS11 1771   | clonality: monoclonal  |  host: mouse  |  reactivity:His-tag

PRODUCT INFORMATION IN PDF

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AS11 1771

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product information
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.

Immunogen

KLH-conjugated synthetic peptide 6xHis

Host Mouse
Clonality Monoclonal
Clone IgG2b, clone HIS.H8 / EH158
Purity
Format Lyophilized
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)
Related products

collection of antibodies to various tags

Plant and algal protein extraction buffer

Secondary antibodies

Additional information

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

application information
Recommended dilution

1 : 1000 with standard ECL (WB)

Expected | apparent MW
Confirmed reactivity

6xHis

Predicted reactivity 6xHis
Not reactive in

No confirmed exceptions from predicted reactivity are currently known

Additional information

Antibody is present in 10 mM PBS, pH 7.2

Selected references Häggmark-Månberg et al. (2016). Autoantibody targets in vaccine-associated narcolepsy. Autoimmunity. 2016 Sep;49(6):421-433. Epub 2016 May 20.

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

||| For other applications, usage on species other than stated above or any other questions, please use the LiveChat option or contact us at support@agrisera.com