His Tag Antibody (33D10.D2.G8) [HRP]

Western Blot: His Tag Antibody (33D10.D2.G8) [HRP] [NBP1-42783] - Lane 1: 100 ng Purified histidine-tagged recombinant protein. Lane 2: 200 ng E. coli cell lysate containing histidine-tagged expression construct. Lane 3: 100 ng Purified GST-tagged recombinant protein. Lane 4: 100 ng Purified FLAG-tagged recombinant protein. Primary antibody: Mouse anti-6xHIS Tag antibody at 1:5,000 overnight at 4C. Secondary antibody: Peroxidase mouse secondary antibody at 1:20,000 for 30 min at RT. Block: 5% BLOTTO for 1 hr at RT.

ELISA: His Tag Antibody (33D10.D2.G8) [HRP] [NBP1-42783] - ELISA of Mouse anti-6xHIS Tag Antibody. Antigen: HIS-tagged purified protein and E. coli cell lysates expressing HIS-Tagged construct, GST- and RON-tagged purified proteins. Coating amount: 0.15 ug per well. Primary antibody: 6xHIS Tag antibody at 100 ug/mL. Dilution series: 2-fold. Mid-point concentration: 200 ng/mL. Secondary antibody: Peroxidase mouse secondary antibody at 1:10,000.

ELISA: His Tag Antibody (33D10.D2.G8) [HRP] [NBP1-42783] - Antigen: 6X HIS-tagged conjugated BSA at the N or C terminus of the 6XHis. Coating amount: 0.15 ug per well. Primary antibody (direct detection): HRP conjugated 6xHIS Tag antibody diluted from stock concentration at 100 ug/mL. Substrate: TMB ELISA peroxidase substrate.

• Reactivity: ET
• Applications: WB, ELISA, IHC
• Clone: 33D10.D2.G8
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: HRP
• Concentration: LYOPH

His Tag Antibody (33D10.D2.G8) [HRP] Summary

• Description: Store vial at 4C prior to restoration. For extended storage aliquot contents and freeze at -20C or below. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4C as an undiluted liquid. Dilute only prior to immediate use.
  
  This protein-A purified antibody is directed against the 6X His motif and is useful in determining its presence in various assays
• Immunogen: His Tag Antibody (33D10.D2.G8) was produced in mice by repeated immunizations with 6X His epitope tag peptide H-H-H-H-H-H conjugated to KLH using maleimide.
• Specificity: This protein-A purified antibody is directed against the 6X His motif and is useful in determining its presence in various assays. This monoclonal anti-6X His tag antibody detects over-expressed proteins containing the 6X His epitope tag. To date, this antibody has reacted with all His tagged proteins so far tested. In western blotting of bacterial extracts, the antibody does not cross-react with endogenous proteins. The antibody recognizes the His-tag (His-His-His-His-His-His) fused to either the amino- or carboxy-termini of targeted proteins in transfected or transformed cells.
• Isotype: IgG1 Kappa
• Clonality: Monoclonal
• Host: Mouse
• Purity: Protein A purified

Applications/Dilutions

• Dilutions:
  • ELISA 1:10000
  • Immunohistochemistry 1:500 - 1:2500
  • Immunohistochemistry-Paraffin 1:10 - 1:500
  • Western Blot 1:1000 - 1:5000
• Application Notes: This product is optimally suited for monitoring expression of His-tagged fusion proteins. As such, anti-6X His/6X His can be used to identify fusion proteins containing the 6X His epitope. The antibody recognizes the His tag fused either to the amino- or carboxy- termini of targeted proteins. This antibody has been tested by ELISA and western blotting against both the immunizing peptide and His-containing recombinant proteins. Although not tested, this antibody is likely functional for immunoprecipitation and immunocytochemistry.
  
  NOTE: Do NOT add Sodium Azide (it inhibits HRP irreversibly).
• Readout System:
  • NovaLume Plus Chemiluminescent Reagent LUM-555

Reactivity Notes

6x His tag specific

Packaging, Storage & Formulations

• Storage: Store lyophilized antibody at 4C in the dark. Aliquot reconstituted liquid and store at -20C. Avoid freeze-thaw cycles.
• Buffer: Lyophilized from 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 10 mg/mL Bovine Serum Albumin (BSA) - Immunoglobulin and Protease free
• Preservative: 0.01% Gentamicin Sulfate
• Concentration: LYOPH
• Purity: Protein A purified
• Reconstitution Instructions: Reconstitute with 100 ul deionized water (or equivalent)

Alternate Names for His Tag Antibody (33D10.D2.G8) [HRP]

• 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 (NBP1-42783)(1)

Publication using NBP1-42783

• Brandsma ME, Diao H, Wang X et al. Plant-derived recombinant human serum transferrin demonstrates multiple functions. Plant Biotechnol J 2010-05-01 [PMID: 20432512]

FAQs for His Tag Antibody (NBP1-42783). (Showing 1 - 1 of 1 FAQ).

1. I'm planning to use the 6-Histidine Epitope Tag Antibody for immunohistochemistry (formalin fixed, paraffin embedded) and am looking for a suitable positive control. Does Novus supply or recommend any positive controls which would be suitable?
   • This antibody is to the epitope tag, so there really isn't anything we can offer as a positive control. Any sample confirmed to have the His-tag would probably be your best bet as a positive control.