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MAb Anti-6HIS Tb cryptate Gold HTRF®

Tb cryptate-labeled anti-6His antibody for capturing 6His-tagged proteins in protein/protein interaction assays.
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  • Ease-of-use Ease-of-use
  • High affinity High affinity
Tb cryptate-labeled anti-6His antibody for capturing 6His-tagged proteins in protein/protein interaction assays.
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Overview

MAb Anti-6HIS-Tb cryptate Gold is a monoclonal antibody which was raised against a polyhistidine tagged fusion protein labeled with Tb. It recognizes synthetic polyhistidine or polyhistidine-tagged fusion protein.

This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, protein/small molecule, receptor/ligand.

HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.

Benefits

  • LARGE COMPLEXES DETECTION
  • BATCH-TO-BATCH REPRODUCIBILITY
  • LOW TO HIGH KD DETECTION

Assay principle

In an HTRF interaction assay, one partner is labeled (directly or indirectly) with the donor, and the other with the acceptor (again, directly or indirectly). The intensity of the signal is proportional to the binding of the 2 partners. In the example shown here: MAb Anti-6HIS Tb cryptate Gold binds to the 6 His tagged partner A while partner B* binds to a specific Ab labeled with an HTRF acceptor. *partner B can also be biotinylated, tagged, Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with acceptor for the detection.
assay principle of detection assay using HTRF MAb Anti-6HIS Tb cryptate Gold

Assay protocol

The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a 6 His-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti-6HIS Tb cryptate Gold (5 µL) and anti-partner B labeled with acceptor (5 µL), incubate and read. *partner B can also be biotinylated, tagged, Fc fused or directly labeled. In these cases, use the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin) labeled with acceptor for the detection.
assay protocol of detection assay using HTRF MAb Anti-6HIS Tb cryptate Gold

How do the number of tests relate to active moiety?

The average conjugate quantity per well reflects overall biological material content. Using the active moiety amount is generally preferred to the quantity of total conjugate. For Cryptate and d2 conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. This is not the case for XL665 labeled entities for which the quantity of total conjugate will vary depending on the final molar ratio of the XL665 conjugate, however, the amount of active moiety, provided by Cisbio, is constant and based on the number of tests ordered.
description of the active moiety in an HTRF conjugate

Recommended quantities of Cryptate and XL665 conjugates

Cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a cryptate concentration of 1 to 5nM is appropriate, and will generate 20,000 to 80,000 cps at 620 nm depending on the HTRF compatible reader used. The XL665 conjugate must match its assay counterpart as closely as possible in order for the maximum number of biomolecules to be tagged with the XL665 acceptor. Thus, to detect a tagged molecule at an assay concentration of 20nM, the concentration of anti-Tag-XL665 should be equimolar or higher.

Europium versus Terbium antibodies to qualify molecular interactions

Depending on the Tag-location the assay performance may vary from one protein to another. Here we describe the parallel use of different Cisbio anti 6His conjugates (Eu, Tb , Eu Gold, Tb Gold) and Lanthascreen anti His tag antibody (Eu, Tb). These cases reveal that depending on the interaction studied, the antibody used might need to be different: in one case the Eu based antibody performs well and in the other, terbium showed better results. For each interaction tested below, the Cisbio Anti6His Gold conjugate out-performed the Lanthascreen Anti-6His tag antibody.

Assay principle of BRD4(1)-H4 peptide interaction

6His-tagged BRD4(1) reader domain binding to the biotinylated peptide substrate H4 is detected by a conjugate mix: donor Cryptate labeled antibody raised against the 6His tag and SA-XL665, for the detection of the biotin-peptide.
Assay principle of BRD1(1)-H4 peptide interaction

Determination of the optimal H4 peptide concentration

The first step of peptide titration revealed that no detection was possible when using Lanthascreen Eu or Tb antibody donor s (data not shown for Tb labeling). For these, no further compound screening can be considered. However, both Gold and the classical version of anti-6His antibodies from Cisbio enabled detection of the interaction. The assay window was increased using the Gold version.
Determination of H4 peptide concentration in BRD4-H4 interaction

JQ1 inhibitory effect on BRD4-H4 interaction

JQ1 inhibitor IC50 was found between 12 and 24.5nM ,in agreement with the literature. As expected, the new Anti-6His cryptate Gold showed an increased assay window .
JQ1 inhibitory effect on BRD4-H4 interaction

Assay principle of NEFcore/ Hck(SH3 domain) prot-prot interaction

NEFcore – HcK SH3 domain interaction is an important target for HIV infectious disease. Inhibitors of this interaction are important targets and are considered as therapeutic solutions. NEFcore , fused to the GST tag is detected using Cisbio PPI MAb Anti GST-d2 antibody at 3 nM. The HcK SH3 domain which contains a 6His tag is detected using the Anti 6His Gold donor conjugate.
Assay principle of NEFcore/ Hck interaction

Determination of optimal GST-Nef concentration

Where no detection was possible when using Cisbio Anti-6His Tb Cryptate (same results observed with Eu, data not shown), the Gold version showed a significant increase in the assay window, enabling further screening of this protein-protein interaction. The results were superior to those obtained using Lanthascreen Tb anti-HIS-tag antibody (same conclusion for Eu, data not shown).
Determination of NEFcore concentration in NEFcore/ Hck interaction

FynSH3 inhibitory effect on NEFcore/ Hck(SH3 domain) interaction

Inhibitor screening assay is shown using FynSH3 protein, which binds to the Nef Core domain competing with the HcK SH3 domain. This showed an IC50 of 0.7-2 µM. The assay window was slightly better using Cisbio Anti-6His Tb cryptate.
FynSH3 inhibitory effect on NEFcore/ Hck interaction

HTRF PPI your dream assay served on a plate

Sandwiches aren't just for eating - Infographics

Best practices for pharmacological characterization of PPI inhibitors

Easy pharmacological characterization of PPI modulators. - Technical Notes

HTRF assays handle low- to high affinity protein-protein interactions

Deciphering low- and high affinity interactions - Application Notes

Nuclear receptor ligand identification with HTRF

Monitoring nuclear receptor binding with HTRF assays - Application Notes

HTRF addresses large protein-protein interaction complexes

Challenge large complexes with HTRF assays - Application Notes

A brief history of Protein-Protein Interactions

How well do you know PPI? - Infographics

Product Insert Anti 6HIS-Tb Gold / 61HI2TLF-61HI2TLA-61HI2TLB

61HI2TLF-61HI2TLA-61HI2TLB - Product Insert

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Virology research solutions using HTRF Protein-Protein Interaction assays

See how peer researchers challenge the viral life cycle with PPI assays - Application Notes

Solutions for CAR-T research

Advance your CAR-T cell research - Flyers

Batch Information Anti 6HIS-Tb Gold / 20230831

61HI2TLA Batch 16A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20240607

61HI2TLA Batch 18A - Batch Information

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61HI2TLA Batch 17A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20250112

61HI2TLA Batch 19A - Batch Information

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61HI2TLA Batch 20A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20221010

61HI2TLB Batch 13A - Batch Information

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61HI2TLB Batch 15A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20230831

61HI2TLB Batch 16A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20240607

61HI2TLB Batch 18A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20240331

61HI2TLB Batch 17A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20250112

61HI2TLB Batch 19A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20250330

61HI2TLB Batch 20A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20220901

61HI2TLF Batch 09A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20240607

61HI2TLF Batch 18A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20240331

61HI2TLF Batch 17A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20250112

61HI2TLF Batch 19A - Batch Information

Batch Information Anti 6HIS-Tb Gold / 20250330

61HI2TLF Batch 20A - Batch Information

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