Key Product Details

Species Reactivity

Epitope Tag

Applications

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, ELISA

Label

HRP

Antibody Source

Monoclonal Mouse IgG1 kappa Clone # 33D10.D2.G8
View all His Tag Primary Antibodies »

Product Specifications

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.

Reactivity Notes

6x His tag specific

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.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1 kappa

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

Scientific Data Images for 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.

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

Application
Recommended Usage

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).

Formulation, Preparation, and Storage

Purification

Protein A purified

Reconstitution

Reconstitute with 100 ul deionized water (or equivalent)

Formulation

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 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store lyophilized antibody at 4C in the dark. Aliquot reconstituted liquid and store at -20C. Avoid freeze-thaw cycles.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: His Tag

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

Long Name

Histidine Tag

Alternate Names

polyHistidine

Additional His Tag Products

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

Certificate of Analysis

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Product Specific Notices for His Tag Antibody (33D10.D2.G8) [HRP]

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.

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

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Protocols

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

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

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  • Q: 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?

    A: 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.

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