His Tag Antibody (2A8)

Product Discontinued

His Tag Antibody (2A8) Summary

• Immunogen: 6x His synthetic peptide coupled to KLH
• Specificity: His-Tag (2A8) Mouse Monoclonal Antibody recognizes over-expressed or recombinant proteins containing the 6x His epitope tag.
• Isotype: IgG1
• Clonality: Monoclonal
• Host: Mouse
• Purity: Protein A or G purified

Applications/Dilutions

• Dilutions:
  • ELISA 1:2000
  • Immunocytochemistry/ Immunofluorescence 1:2000
  • Immunoprecipitation 1:100
  • Western Blot 1:5000

Reactivity Notes

6-His Tagged Proteins

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS (pH 7.4)
• Preservative: 0.05% Sodium Azide
• Concentration: 1 mg/ml
• Purity: Protein A or G purified

Alternate Names for His Tag Antibody (2A8)

• 6 His epitope tag
• 6-His Tag
• 6X His Tag
• 6X His
• 6x-His Tag
• 6X-His
• H
• Hexa His tag
• HHHHHH epitope tag
• HHHHHH tag
• His Tag
• HIS
• His6-Tag
• polyhistidine tag
• polyHistidine
• Poly-histidine

Background

A his-tag (also known as histidine tag or polyhistidine tag) is a common epitope tag that typically consists of at least 6 histidine residues fused to either the carboxyl (C-) or amino (N-) terminus of a targeted recombinant protein to facilitate its purification and detection (1). The most common his-tag is the hexahistidine (His6/6-His) tag which has a theoretical molecular weight of 0.8kda (1). The histidine residues readily interact with transition metal ions such as Co2+, Ni2+, Cu2+, and Zn2+, making immobilized metal-affinity chromatography (IMAC) the preferred technique for his-tag purification (1, 2). Metal ions are immobilized and bound to by His-tags in the IMAC column via the histidine imidazole ring. The tagged protein can be eluted off the column by washing with buffers containing a low concentration of imidazole (1, 2). Due to its relatively small size, low immunogenicity, versatility under denaturing conditions, and minimal interference with the structure and function of proteins, the his-tag is one of the most widely used tags for protein purification (1-3).

References

1. Malhotra, A. (2009). Tagging for protein expression. Methods in Enzymology, Guide to Protein Purification, 2nd Edition, 463, 239-258. https://doi.org/10.1016/s0076-6879(09)63016-0

2. Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60(5), 523-533. https://doi.org/10.1007/s00253-002-1158-6

3. Booth, W. T., Schlachter, C. R., Pote, S., Ussin, N., Mank, N. J., Klapper, V., ... Chruszcz, M. (2018). Impact of an N-terminal polyhistidine tag on protein thermal stability. ACS Omega, 3(1), 760-768. https://doi.org/10.1021/acsomega.7b01598

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for His Tag Antibody (NBP1-41288)(1)

Publication using NBP1-41288

• Chen KY, Santos Afonso ED, Enouf V et al. Influenza virus polymerase subunits co-evolve to ensure proper levels of dimerization of the heterotrimer PLoS Pathog. 2019-10-01 [PMID: 31581279] (WB)

FAQs for His Tag Antibody (NBP1-41288). (Showing 1 - 4 of 4 FAQ).

1. I would like to know if it is possible to use 6-Histidine Epitope Tag Antibody (2A8) to purify the His-contained protein?
   • It is possible to purify a HIS-tagged protein using a HIS tag antibody. We unfortunately do not perform this type of purification at our site, so therefore I do not have a protocol available. For a protocol, I suggest consulting the scientific literature to find one that will apply to your experiment.
2. Could you please give me the protocol used to obtain the nice published result related to t 6-Histidine Epitope Tag Antibody (2A8) (NBP1-41288)?
   • The western blot images for this product were obtained using over-expressed His-tagged protein in 293T cell lysate (first image) and 1 ng of 6-His epitope recombinant protein (second image). We followed our general western blot procedure outlined at the following link: Western-blot Protocol.
3. Could you please give me the dilution of the antibodies that has been used? My protocol is similar to yours but I have a pronounced background staining. Any specific suggestions?
   • We are using the antibodies at 1:5000. This is a starting recommendation, and one based on use with over-expression lysates and the recombinant protein. Most of the time, uniform background staining can be solved with proper blocking. Please be sure to block your membrane for at least one hour and up to 3 hours prior to the primary antibody incubation. We use 5%NFDM and 1%BSA in TBST to block our membranes. You may also want to run a secondary only test, to make sure your secondary is not unspecifically binding to the membrane. Another suggestion is to increase your washes or wash more vigorously. We wash our blots 5 x 5 minutes each on a flat shaker on high.
4. Could you tell me if this antibody will detect a his tag on a protein with an amino acid sequence preceding the 6x hisidine of GGS? Or following the 6x his tag with amino acid sequence "SGG"?
   • NBP1-41288 has been tested against tags both N and C-terminal. A few preceding amino acids should not interfere with this tag. If the tag is internal, there may be some interference; however, we have not yet tested that with this product.