IKP

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

Product no: AS11 1771

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

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  • Product Info
  • Immunogen:

    KLH-conjugated synthetic peptide 6xHis

    Sub class: IgG2b
    Host: Mouse
    Clonality: Monoclonal
    Purity: Total IgG fraction. Protein A purified.
    Format: Liquid
    Quantity: 50 µg
    Storage: Store at -20C.
    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
  •  

    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 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.

    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
    Additional information (application):

    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
  • 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: De Brasi-Velasco et al. (2021). Autophagy Is Involved in the Viability of Overexpressing Thioredoxin o1 Tobacco BY-2 Cells under Oxidative Conditions. Antioxidants. 2021; 10(12):1884. https://doi.org/10.3390/antiox10121884
    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.
    López-Vidal et al. (2020). Is Autophagy Involved in Pepper Fruit Ripening? Cells, 9 (1) , DOI: 10.3390/cells9010106
    Häggmark-Månberg et al. (2016). Autoantibody targets in vaccine-associated narcolepsy. Autoimmunity. 2016 Sep;49(6):421-433. Epub 2016 May 20.
  • Protocols
  • Antibody protocols

  • Reviews:
  • To leave a review, you should have previously purchased this product and to your customer profile.

    HIM Sok Lay | 2021-02-10
    0 | 0
    The antibody worked very well with the western blot. Im happy with the results.

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