Anti-6X His tag® antibody [4D11] (ab5000)

Reviews (4)Q&A (6)References (17)

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
  • Specificity
    This antibody recognises all proteins which contain either an N or C-terminal HHHHHHGS epitope tag. It has been shown by a reviewer to recognise HHHHHH alone.
  • Tested applications
    Suitable for: Dot blot, ELISA, WB, IP, ICC/IF, Other, Flow Cytmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Recombinant malaria protein TRAP fused at the amino-terminal end with six His tag. The coding sequence of TRAP 1.0 was amplified from P. falciparum genomic DNA (ITO4 isolate) in standard Polymerase Chain Reaction PCR (Saiki et al. 1988) using the primer combination trap 1 5'-AGTTGGATCCAGAGATGTGCAAAACA ATATAGTGG-3' and trap 2 5'-GCGAGTAAAGCTAAGCTTCCAGCTATTCCACCTGC-3', the amplified DNA sequence was cloned in the vector pDS56/RBSII,6xHis (Hochuli et al. 1988).

  • 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
  • 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

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.
Other 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

References

This product has been referenced in:
See all 17 Publications for this product

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

Customer reviews and Q&As

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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!

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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

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
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

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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?

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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?

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

 Excellent
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
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
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
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?

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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.

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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
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

Question

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

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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.

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