QIAexpress Detection Systems are designed for simple and sensitive detection of 6xHis-tagged proteins. QIAexpress detection reagents include high-affinity, high-specificity monoclonal Anti·His Antibodies and antibody conjugates, and Ni-NTA Conjugates. The reagents have been developed to allow detection of almost any 6xHis-tagged protein. The 6xHis Protein Ladder is the ideal positive control and molecular weight standard for western blotting using the 6xHis detection reagents. In addition, the Tag·100 Antibody is provided for detection of proteins with the Tag·100 epitope expressed from the pQE-100 DoubleTag Vector. Proteins encoded by this vector contain a 6xHis tag at the N-Terminus allowing directed immobilization.
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n.a.: not applicable; n.d.: not determined |
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Recombinant proteins expressed from pQE-9, pQE-30, pQE-31, pQE-32, pQE-40, pQE-80L, pQE-81L, pQE-82L, pQE-100 DoubleTag, pRSET (Invitrogen), and pBlueBacHis (Invitrogen) encode this epitope. |
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Detection using chemiluminescent substrate. |
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Detection using alkaline phosphatase chromogenic substrate. The apparent sensitivity on western blots is lower than on dot blots due to loss of protein during transfer. |
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Anti·His HRP Conjugates are available as Penta·His, Tetra·His, and RGS·His variants, and have the same 6xHis tag recognition and binding properties as the corresponding antibody. |
Detection of 6xHis-tagged Proteins with QIAexpress 6xHis Detection Systems
Principle
QIAexpress
Anti·His Antibodies comprise three highly
sensitive monoclonal antibodies that allow detection of almost any protein
possessing accessible stretches of consecutive histidine
residues. The 6xHis tag may be located at the N-terminus, at the C-terminus, or
internally. In some cases, even partially hidden 6xHis tags can be detected.
QIAexpress Anti·His Antibodies display a markedly higher sensitivity for 6xHis
tags than other commercially available antibodies (1). To
determine which antibody is optimal for your specific protein and application,
QIAGEN offers the Anti·His
Antibody Selector Kit containing 3 µg of each of the three antibodies.
New Anti·His HRP Conjugates consist of mouse monoclonal IgG1 Anti·His Antibodies coupled to horseradish peroxidase. They can be used for direct detection of any protein with an accessible 6xHis tag, by chromogenic or chemiluminescent methods. They eliminate the need for secondary antibodies, saving time and expense in blotting and ELISA procedures. The conjugates are available in highly specific Penta·His, Tetra·His, and RGS·His forms (see QIAexpress 6xHis Detection Reagent Selection Guide). Anti·His HRP Conjugates are supplied with an optimized Blocking Reagent and Blocking Reagent Buffer to ensure optimal specificity and sensitivity.
New Penta·His Alexa Fluor Conjugates allow highly sensitive and specific direct detection of 6xHis-tagged proteins in immunofluorescence procedures. The Alexa Fluor dyes conjugated to Penta·His antibodies cover a wide portion of the visible spectrum, are photostable, and fluoresce intensely over a wide pH range.
New Penta·His Biotin Conjugates consist of Penta·His Antibodies conjugated to biotin. They can be used in conjunction with any avidin or streptavidin detection conjugate allowing colorimetric, chromogenic, chemiluminescent, or fluorescent detection of 6xHis-tagged proteins.
New Streptavidin-R-PE is used in conjunction with Penta·His Biotin Conjugates for highly sensitive fluorescent detection of 6xHis-tagged proteins.
Ni-NTA Conjugates consist of Ni-NTA coupled to calf intestinal alkaline phosphatase (AP) or horseradish peroxidase (HRP). They can be used for chromogenic detection of any recombinant protein containing an accessible histidine tag — without the use of secondary antibodies. Detection using Ni-NTA Conjugates is based on the same Ni-NTA technology that underlies the purification and assay of 6xHis-tagged biomolecules (see QIAexpress — The Complete System; Ni-NTA Technology and the 6xHis Tag ). The Ni-NTA ligand conjugated to a reporter enzyme binds to the histidine residues in the 6xHis tag region. The location of the bound enzyme-conjugated Ni-NTA ligand is revealed by adding enzyme substrates that produce colored reaction products.
The Tag·100 Antibody is a powerful assay-design tool which enables detection of proteins that have the Tag·100 epitope expressed from the pQE-100 DoubleTag Vector, which also encodes the 6xHis tag. The presence of two tags, one at each end of the protein, allows specific capture using Ni-NTA HisSorb Strips or Plates or Ni-NTA Agarose Magnetic Beads and specific detection using the Tag·100 Antibody.
The 6xHis Protein Ladder serves as a molecular weight standard and a positive control for western blotting using QIAexpress Detection Systems. It consists of five 6xHistagged proteins in convenient sizes ranging from 15 to 100 kDa.
QIAexpress Detection Systems allow efficient detection of 6xHis-tagged proteins and are ideal for many applications including:
QIAexpress Anti·His Antibodies and conjugates have many additional useful applications including:
Immunohistochemistry with Penta·His Antibody
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Immunolocalization of 6xHis-tagged β-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. |
6xHis-Tagged Thioredoxin Detected by Penta·His Antibody in Yeast Cells
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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. |
Identification of Positive Expression Clones by Colony Blotting
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Colony blot detection of positive expression clones with Penta·His Antibody. A BamHI–SalI 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. |
Sensitivity of QIAexpress Anti·His Antibodies
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Detection of 6xHis-tagged proteins (A–H, see table "Proteins tested with QIAexpress Anti·His Antibodies") 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. |
Proteins tested with QIAexpress Anti·His Antibodies
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Cited Reference
1. Monoclonal anti-His antibodies for sensitive detection of 6xHis-tagged proteins. QIAGEN News 1997 No. 3, 1.
For a list of references citing QIAexpress products for detection of 6xHis-tagged proteins, visit the QIAGEN Reference Database online at www.qiagen.com/RefDB/seach.asp or contact QIAGEN Technical Services or your local distributor.
