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Anti·His Antibody Selector Kit

For cost-effective comparison and determination of the optimal detection antibody for His-tagged proteins

  • Cost-effective determination of optimal antibody
  • Three Anti·His Antibodies to try with new proteins/applications
  • Detection of C-terminal, N-terminal, and internal His tags
  • Highly specific and sensitive detection
  • Negligible background signals in cell extracts
Monoclonal mouse Anti·His Antibodies (IgG1) are used to detect recombinant proteins carrying His tags. Three antibodies are included (RGS·His, Penta·His, and Tetra·His), each 3 µg as BSA-free lyophilized powder, providing a 30 ml working solution. Each antibody detects a different His-tag epitope, with N-terminal, C-terminal, and internal His tags being detected by both Penta·His and Tetra·His antibodies, while RGS·His antibody is restricted to the N-terminus with a RGS-His tag. The antibodies have negligible cross-reactivity with crude E. coli, yeast, insect, or mammalian cell lysates.
Cat No./ID: 34698
Anti·His Antibody Selector Kit
$175.00
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RGS·His Antibody, Penta·His Antibody, Tetra·His Antibody, all BSA-free, 3 µg each
The Anti·His Antibody Selector Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Detection of 6xHis-tagged thioredoxin in yeast cells.
The indicated amounts of purified 6xHis-tagged thioredoxin mixed with crude extract from yeast cells (5 µg total protein per lane) were applied to a 15% SDS-polyacrylamide gel. After electrophoresis and western transfer, 6xHis-tagged proteins were detected with Penta·His Antibody followed by chemiluminescent detection with HRP-conjugated rabbit anti-mouse IgG and ECL detection reagent. 6xHPL: 6xHis Protein Ladder.
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Sensitivity of QIAexpress Anti·His Antibodies.

Detection of 6xHis-tagged proteins with QIAexpress Anti·His Antibodies (A: DHFR; B: DHFR; C: thioredoxin; D: TNF-α; E: TFIIAγ; F: chaperonin; G DNA polymerase; H: TFIIAαß; for tag location and sequences detected see table "Proteins detected with QIAexpress Anti·His Antibodies"). Indicated amounts of pure 6xHis-tagged protein were applied to a nitrocellulose membrane, and detection was carried out with the Anti·His primary antibody indicated diluted 1/2000, followed by chromogenic detection with AP-conjugated rabbit anti-mouse IgG and NBT/BCIP.

b

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Identification of positive expression clones by colony blotting.
Colony blot detection of positive expression clones with Penta·His Antibody. A BamHI–Sal I fragment encoding a DNA polymerase was cloned into pQE-31, transformed into E. coli M15[pREP4], grown overnight at 37°C, replica-plated onto a nitrocellulose membrane, and treated according to the protocol in The QIAexpressionist with NBT/BCIP detection. Colonies of positive expression clones appear much darker than negative clones.
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Immunohistochemistry with Penta·His Antibody.

Immunolocalization of 6xHis-tagged ß-galactosidase in murine NIH/3T3 fibroblast cells. Following transfection, tagged protein was detected using Penta·His Antibody followed by FITC/conjugated goat anti-mouse IgG antibody and visualized by fluorescence microscopy.

Performance

The sensitivity of the His Antibodies in western blots is up to 2 ng, and up to 0.5 ng in dot blots (see Figure "Sensitivity of QIAexpress Anti·His Antibodies"), when carrying out detection using a chromogenic substrate (sensitivity may vary with different proteins).

 

Proteins detected with QIAexpress Anti∙His Antibodies.
    Protein  Tag location Sequence
A     DHFR  N-terminus                                MRGSHHHHHHGSVRPLNCIV..........
B     DHFR  C-terminus            .........VYEKKDGSRSHHHHHH
C     Thioredoxin  N-terminus                                MRGSHHHHHHGSSDKIIHLT..........
D     TNF-α  N-terminus                                    MRHHHHHHQSTESMIRDV.........
E     TFIIAγ  N-terminus                                MGSSHHHHHHSSGLVPRGSH.....
F     Chaperonin  C-terminus            ..........AKIAKDGSRSHHHHHH
G

    DNA polymerase

 C-terminus            ..........EDWLSAKERSHHHHHH
H

    TFIIAαß

 Internal            ......QQQHQPQQQQHHHHHHHQQAQPQQTVP....

Principle

All QIAexpress mouse monoclonal antibodies are produced under conditions that guarantee high purity and activity. Hybridoma cell culture is carried out under serum-free conditions, ensuring that the preparations are free of viruses, mycoplasma, and contaminating immunoglobulins. Purification by adsorption chromatography is performed entirely at physiological pH, yielding preparations with the highest proportion of native antibody. Tetra·His Antibody and Penta·His Antibody can both be used for detecting C-terminal, N-terminal, and internal His-tags. The RGS·His Antibody is restricted to His-tags at the N-terminus of proteins expressed with the RGS-His tag.

QIAexpress Detection Systems allow efficient detection of His-tagged proteins, and are ideal for many applications, including, colony, dot, and western blotting procedures (see figures "Detection of 6xHis-tagged thioredoxin in yeast cells", "Identification of positive expression clones by colony blotting", "Sensitivity of QIAexpress Anti·His Antibodies"), as well as screening for positive expression clones (see figure "Identification of positive expression clones by colony blotting"). QIAexpress Anti·His Antibodies and conjugates have many additional useful applications, including immunoprecipitation, ELISA, immunocytochemistry, immunohistochemistry (see figure "Immunohistochemistry with Penta·His Antibody"), protein localization and targeting studies, and LabMAP bead-based assays.

Applications

QIAexpress Detection Systems allow efficient detection of 6xHis-tagged proteins and are ideal for many applications, including:

  • Colony, dot, and western blotting procedures
  • Screening for positive expression clones
  • Monitoring expression levels and stability of 6xHis-tagged proteins
  • Immunoprecipitation and ELISA
  • Immunocytochemistry and immunohistochemistry
Features
Specifications
Detection Secondary antibody required
Epitope detected HHHH/HHHHH/RGSHHHH
Sensitivity in dot blots -chromogenic substrate- 0.5 ng
Sensitivity in western blots -chromogenic substrate- 2 ng
Substrate for blot detection Dependent on secondary antibody
Substrates for assay procedure Dependent on secondary antibody
Tag His tag

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Handbooks & Protocols (1)
For Anti·His Antibodies, Anti·His HRP Conjugates, Penta·His™ Alexa Fluor® Conjugates, Penta·His Biotin Conjugate, Ni-NTA Conjugates, Tag·100™ Antibody, Streptavidin–R-PE, 6xHis Protein Ladder, Ni-NTA HisSorb™ Strips and Plates 
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Kit Handbooks (1)
For Anti·His Antibodies, Anti·His HRP Conjugates, Penta·His™ Alexa Fluor® Conjugates, Penta·His Biotin Conjugate, Ni-NTA Conjugates, Tag·100™ Antibody, Streptavidin–R-PE, 6xHis Protein Ladder, Ni-NTA HisSorb™ Strips and Plates 
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