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    6x-his-tag-antibody-4d11-ab5000.pdf

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Tags & Cell Markers Epitope Tags His Tag
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Anti-6X His tag® antibody [4D11] (ab5000)

  • Datasheet
  • SDS
Reviews (4)Q&A (6)References (22)

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Western blot - Anti-6X His tag® antibody [4D11] (ab5000)
  • Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody [4D11] (ab5000)
  • Western blot - Anti-6X His tag® antibody [4D11] (ab5000)
  • Immunoprecipitation - Anti-6X His tag® antibody [4D11] (ab5000)

Key features and details

  • Mouse monoclonal [4D11] to 6X His tag®
  • Suitable for: Dot blot, ELISA, WB, IP, ICC/IF, Flow Cyt
  • Reacts with: Species independent
  • Isotype: IgG2a

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Overview

  • Product name

    Anti-6X His tag® antibody [4D11]
    See all 6X His tag® primary antibodies
  • Description

    Mouse monoclonal [4D11] to 6X His tag®
  • Host species

    Mouse
  • Tested applications

    Suitable for: Dot blot, ELISA, WB, IP, ICC/IF, Flow Cytmore details
  • Species reactivity

    Reacts with: Species independent
  • Immunogen

    corresponding to 6X His tag®.

  • Epitope

    This antibody was shown to recognize an epitope (HHHHHHGS) encoded by the polylinker of several expression vectors (including pDS:RBSII, pQE-30, and pQE-50).
  • Positive control

    • Purchase matching WB positive control:Recombinant ENO1 protein
  • General notes

    This antibody clone is manufactured by Abcam.

    HIS-TAG® is a trademark of EMD Biosciences, Inc.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Add glycerol to a final volume of 50% for extra stability and aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: 6.97% L-Arginine, PBS
  • Concentration information loading...
  • Purity

    IgG fraction
  • Clonality

    Monoclonal
  • Clone number

    4D11
  • Myeloma

    NS1
  • Isotype

    IgG2a
  • Light chain type

    kappa
  • Research areas

    • Tags & Cell Markers
    • Epitope Tags
    • His Tag

Associated products

  • Alternative Versions

    • Anti-gamma Tubulin antibody [4D11] - C-terminal (ab173831)
    • Anti-ATP5D antibody [4D11] (ab174438)
    • HRP Anti-6X His tag® antibody [4D11] (ab202004)
    • Anti-hnRNP L antibody [4D11] (ab6106)
    • FITC Anti-LLGL1 antibody [4D11] (ab95802)
  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
  • Isotype control

    • Mouse IgG2a, kappa monoclonal [MG2a-53] - Isotype control (ab18415)

Applications

Our Abpromise guarantee covers the use of ab5000 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
AP Use at an assay dependent concentration.
Dot blot Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
WB 1/1000.
IP Use at an assay dependent concentration. See Abreview.
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

Target

  • Relevance

    The H-H-H-H-H-H motif is used as a tag on many recombinant proteins to facilitate purification. The antibody recognizes the His-tag fused to the amino- or carboxy- termini of targeted proteins in transfected or transformed cells.
  • Cellular localization

    Depends upon the localization of the parent protein tagged with hexahistidine.
  • Alternative names

    • 6 His epitope tag antibody
    • Hexa His tag antibody
    • HHHHHH epitope tag antibody
    • HHHHHH tag antibody
    • His tag antibody
    • Polyhistidine Tag antibody
    see all

Images

  • Western blot - Anti-6X His tag® antibody [4D11] (ab5000)
    Western blot - Anti-6X His tag® antibody [4D11] (ab5000)
    Anti-6X His tag® antibody [4D11] (ab5000) at 1/1000 dilution + 6X His Tagged Protein at 1 µg

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 30 seconds
  • Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody [4D11] (ab5000)
    Immunocytochemistry/ Immunofluorescence - Anti-6X His tag® antibody [4D11] (ab5000)This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic
    ab5000 staining Human HEK 293 cells transfected with CACNB3-His by ICC/IF. Cells were PFA fixed and permeabilized in 0.5% Triton X-100 prior to blocking in 5% serum for 20 minutes at 25°C. The primary antibody (1/250 in 1% goat serum, 0.1% TX100, 1X PBS) was incubated with the sample for 16 hours at 4°C. An Alexa Fluor® 546 conjugated goat anti-mouse antibody (1/500) was used as the secondary.  DAPI was used for nuclei staining (blue).

    See Abreview

  • Western blot - Anti-6X His tag® antibody [4D11] (ab5000)
    Western blot - Anti-6X His tag® antibody [4D11] (ab5000)This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic
    All lanes : Anti-6X His tag® antibody [4D11] (ab5000) at 1/2000 dilution

    Lane 1 : Lysates prepared from HEK-293 cells
    Lane 2 : Lysates prepared from HEK-293 cells transfected with Beta3-His (CACNB3)

    Secondary
    All lanes : HRP-conjugated goat anit-mouse IgG polyclonal at 1/5000 dilution

    See Abreview

  • Immunoprecipitation - Anti-6X His tag® antibody [4D11] (ab5000)
    Immunoprecipitation - Anti-6X His tag® antibody [4D11] (ab5000)This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic

    ab5000 Immunoprecipitate in human HEK293 whole cell lysate. 25µg of cell lysate incubated with primary antibody (undiluted) and matrix (Protein G) for 16 hours at 4°C. For western blotting an undiluted HRP-Goat anti-rabbit IgG polyclonal was used. Beta3-His fusion protein was immunoprecipitated using anti His Ab, and specific beta3a band (56kDa) was detected using anti beta3 Ab

    Lane 1. Lysate of HEK293 cells expressing beta3-His (CACNB3)

    Lane 2. IP with anti His Ab

    Lane 3. Non bound fraction

    See Abreview

Protocols

  • Immunoprecipitation protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

Datasheets and documents

    • Datasheet
    • SDS
  • References (22)

    Publishing research using ab5000? Please let us know so that we can cite the reference in this datasheet.

    ab5000 has been referenced in 22 publications.

    • Lv Z  et al. CD19-targeting fusion protein combined with PD1 antibody enhances anti-tumor immunity in mouse models. Oncoimmunology 9:1747688 (2020). PubMed: 32363119
    • Chen X  et al. A switch in the poly(dC)/RmlB complex regulates bacterial persister formation. Nat Commun 10:27 (2019). PubMed: 30604752
    • Herrera T EA  et al. GlSir2.1 of Giardia lamblia is a NAD+-dependent cytoplasmic deacetylase. Heliyon 5:e01520 (2019). PubMed: 31025022
    • Wang C  et al. Lysyl Oxidase-Like Protein 2 Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer. Neoplasia 21:413-427 (2019). PubMed: 30925417
    • Panagiotakos G  et al. Aberrant calcium channel splicing drives defects in cortical differentiation in Timothy syndrome. Elife 8:N/A (2019). PubMed: 31868578
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 10 Abreviews or Q&A

    Question

    Inquiry: Hello, We are interested in the His Tag antibody for applications including ICC, WB, and IP primarily. Both of the products below seem to be used in this fashion and both have good reviews and are the same price. Could you please suggest or clarify the differences between them? We will have both C-term or N-term fusion proteins that we need to detect in cells. Products: ab18184 vs ab5000 Thank you!

    Read More

    Abcam community

    Verified customer

    Asked on Feb 19 2013

    Answer

    The main difference between ab18184 and ab5000 is the immunogen used to produce the antibodies. ab18184 was produced against a 6X His synthetic peptide, while ab5000 was raised against a recombinant protein fused to a 6X His tag. Thus, ab18184 was originally designed to detect HHHHHH alone while ab5000 was orginally designed to detect HHHHHHGS. A reseacher tested ab5000 and submitted an abreview showing that it also recognizes the HHHHHH alone.

    Read More

    Abcam Scientific Support

    Answered on Feb 19 2013

    Question

    I was looking for an anti-His-tag antibody. I
    found that in the description in some of them (ab5000, ab18184) it is
    indicated that the antibody recognizes recombinant protein expressed
    in mammalian cells and in some other (ab15149, ab81663) it is
    specified that the antibody recognizes protein expressed in bacteria.
    If the antibody is raised against His-tag epitope can it recognize
    indifferently recombinant protein expressed either in bacteria either
    in mammalian or eukariotic cells? or not?
    I had some problem with an anti-His-tag antibody of another company
    since recognize the protein expressed in bacteria but not in mammalian
    in WB and I would like to understand the reason before to look for a
    product from a different company

    Read More

    Abcam community

    Verified customer

    Asked on Nov 20 2012

    Answer

    I can confirm that all four products, the ab5000, ab15149, ab18184 and ab81663 detect proteins, which contain either an N or C-terminal HHHHHHGS (6x) epitope tag.

    Important aspects for his tag recognition are, the localisation of the tag (N- or C-terminal) and the length of the his tag, that should be identical with the antibody chosen.

    Furthermore it is important to confirm that the tagged protein is expressed in transfected or transformed cells. If the recombinant protein is expressed, it should not make a difference for the detection if either a bacteria or a mammalian system was used.

    However, some his tag antibodies could cross react with other proteins in the used system. For ab81663, ab15149 there may be seen some cross reactivity with extracts of insect or mammalian cells

    Read More

    Abcam Scientific Support

    Answered on Nov 20 2012

    Question

    WB seeing one single band, however band is present in control samples with no His tag as well as tagged samples. Control samples do have glycine, can this be cross-reactivity from Glycine?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 13 2012

    Answer

    I really wouldn't expect the antibody would cross-react with glycine, particularly as there is only a single glycine in the epitope tag. If it recognised a single glycine it'd cross react with nearly all untagged proteins as it's a very common amino acid. I can't think of any proteins that have an internal string of histidines but perhaps that is the problem?

    Read More

    Abcam Scientific Support

    Answered on Nov 13 2012

    Immunocytochemistry/ Immunofluorescence abreview for Anti-6X His tag® antibody [4D11]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HEK 293 cells transfected with CACNB3-His)
    Specification
    HEK 293 cells transfected with CACNB3-His
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% TX100
    Blocking step
    Serum as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Herr Dr. Vladimir Milenkovic

    Verified customer

    Submitted Nov 20 2009

    Immunoprecipitation abreview for Anti-6X His tag® antibody [4D11]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunoprecipitation
    Sample
    Human Cell lysate - whole cell (HEK 293 cells transfected with Beta3-His (CACNB3))
    Total protein in input
    25 µg
    Specification
    HEK 293 cells transfected with Beta3-His (CACNB3)
    Immuno-precipitation step
    Protein G
    Read More

    Herr Dr. Vladimir Milenkovic

    Verified customer

    Submitted Nov 20 2009

    Western blot abreview for Anti-6X His tag® antibody [4D11]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK 293 cells transfected with Beta3-His (CACNB3))
    Loading amount
    25 µg
    Specification
    HEK 293 cells transfected with Beta3-His (CACNB3)
    Gel Running Conditions
    Reduced Denaturing (10% SDS)
    Blocking step
    Milk as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Herr Dr. Vladimir Milenkovic

    Verified customer

    Submitted Nov 20 2009

    Western blot abreview for Anti-6X His tag® antibody [4D11]

    Good
    Abreviews
    Abreviews
    Application
    Western blot
    Sample
    Human Recombinant protein (recombinant protein (human))
    Loading amount
    20 µg
    Specification
    recombinant protein (human)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 27 2006

    Question

    We have a protein with C- terminal PMAHHHHHH. This ab5000 is able to recognise it? This product recognise any 6-his tag?

    Read More

    Abcam community

    Verified customer

    Asked on Jan 24 2006

    Answer

    This antibody does indeed recognise the classical 6 x His tag HHHHHH, and therefore I would fully expect it to recognise your PMAHHHHHH motif.

    Read More

    Abcam Scientific Support

    Answered on Jan 26 2006

    Question

    Our proteins are labled with the following tag: N-RGSHHHHHH-C Thus in the case of our lable a serine residue precedes the His sextet. The ab5000 epitope is presented as recognizing HHHHHHGS, thus regardless of whether the tag is C or N terminal, the His sextet is neighbored directly by Glycine instead of serine. I would appreciate verification that our epitope will be recognized by ab5000 before investing in this antibody.

    Read More

    Abcam community

    Verified customer

    Asked on Oct 06 2004

    Answer

    The immunogen of this antibody is recombinant malaria protein TRAP fused at the amino-terminal end with six his tag. It does recognize proteins which contain either an N or C-terminal HHHHHHGS epitope tag. Unfortunately, we do not know how neighbouring glycine would influence the binding of the antibody to the epitope.

    Read More

    Abcam Scientific Support

    Answered on Oct 07 2004

    Question

    Hi, For the His tag antibody (4D11), what is the protein concentration? Thanks, Ana

    Read More

    Abcam community

    Verified customer

    Asked on Oct 13 2003

    Answer

    We are only able to guarentee the concentration of purified antibodies, and as this antibody is ascitic fluid it does not fall into that catagory.

    Read More

    Asdf Edo

    Abcam Scientific Support

    Answered on Oct 13 2003

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