His Tag Antibody (33D10.D2.G8) [DyLight 488]

Western Blot: His Tag Antibody (33D10.D2.G8) [DyLight 488] [NBP1-97329] - Western Blot of His Tag antibody (33D10.D2.G8) [DyLight 488].Lane 1: 100ng Purified histidine-tagged recombinant protein.Lane 2: 200ng E. coli cell lysate containing histidine-tagged expression construct.Lane 3: 100ng Purified GST-tagged recombinant protein.Lane 4: 100ng Purified FLAG-tagged recombinant protein.Primary antibody: Mouse anti-His 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.

Immunocytochemistry/Immunofluorescence: His Tag Antibody (33D10.D2.G8) [DyLight 488] [NBP1-97329] - DyLight(TM) dyes can be used for multi-color immunofluorescence microscopy with uniform fluorescence intensity throughout the image. DyLight(TM) dyes are exceptionally bright and photostable and are optimized for microscopy and microarray detection methods. This image shows anti-histone detection using a DyLight(TM) 488 conjugate (green). Anti-Tubulin was detected using a DyLight(TM) 549 conjugate (red). Nuclei were counter-stained using DAPI (blue). The image was captured using an Axio Imager.Z1 (Zeiss Micro Imaging Inc).

Immunocytochemistry/Immunofluorescence: His Tag Antibody (33D10.D2.G8) [DyLight 488] [NBP1-97329]

Fluorophore-linked immunosorbent assay: His Tag Antibody (33D10.D2.G8) [DyLight 488] [NBP1-97329]

ELISA: His Tag Antibody (33D10.D2.G8) [DyLight 488] [NBP1-97329] - ELISA of His Tag antibody (33D10.D2.G8) [DyLight 488].Antigen: HIS-tagged purified protein and E. coli cell lysates expressing HIS-Tagged construct, GST- and RON-tagged purified proteins.Coating amount: 0.15ug per welll.Primary antibody: His Tag antibody at 100ug/mL.Dilution series: 2-fold.Mid-point concentration: 200ng/mL.Secondary antibody: Peroxidase mouse secondary antibody at 1:10,000.Substrate: TMB.

• Reactivity: ET
• Applications: WB, DB, ELISA, ICC/IF
• Clone: 33D10.D2.G8
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: DyLight 488
• Concentration: LYOPH

His Tag Antibody (33D10.D2.G8) [DyLight 488] 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:
  • Dot Blot
  • ELISA
  • Fluorophore-linked immunosorbent assay >1:20000
  • Immunocytochemistry/ Immunofluorescence >1:5000
  • Western Blot 1:10000 - 1:25000
• Application Notes: This product has been tested by ELISA and western blot. The emission spectra for this DyLight(TM) conjugate match the principle output wavelengths of most common fluorescence instrumentation.

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% Sodium Azide
• Concentration: LYOPH
• Purity: Protein A purified
• Reconstitution Instructions: Reconstitute with 100 ul deionized water (or equivalent)

Notes

DyLight (R) is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.

Alternate Names for His Tag Antibody (33D10.D2.G8) [DyLight 488]

• 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.