Eu cryptate-labeled anti-6His antibody for capturing 6His-tagged proteins in protein/protein interaction assays.
See more
  • No-wash
  • Ease-of-use
  • High affinity
Eu cryptate-labeled anti-6His antibody for capturing 6His-tagged proteins in protein/protein interaction assays.
-
In stock

Overview

MAb Anti-6HIS-Eu cryptate is an IgG2a raised against a polyhistidine tagged fusion protein labeled with Eu. 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.

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-Eu cryptate binds to the 6 His tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *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 donor for the detection.

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-Eu cryptate (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.

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.

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.

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.

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 .

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.

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).

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.
Applications of HTRF and Tag-lite-lite Assays for HTP Antibody Screening

In collaboration with Bristol Myers Squibb - Scientific Presentations

Academia/Industry Cross-Fertilization through Translational Research

In collaboration with Scripps - Scientific Presentations

HTRF-powered detection of ubiquitin-like protein (UBL) signaling and interactions

In collaboration with Sanford/Burnham - Scientific Presentations

The use of HTRF in biologics discovery

In collaboration with MedImmune - Scientific Presentations

HTRF: One technology, many uses in a pre-clinical laboratory setting

Benefits and considerations of HTRF - Scientific Presentations

Exploiting HTRF for novel drug classes: Stabilizing 14-3-3 protein-protein interactions

In collaboration with Lead Discovery Center GmbH - Scientific Presentations

HTRF a versatil approach for 7TM drug discovery

In collaboration with MRC - Scientific Presentations

HTRF protein-protein interaction reagents

Benefit from unlimited flexibility for your assay development - Flyers

Addressing the interactome with protein-protein assays

Cover all PPIs with one approach - Brochures

HTRF -A Beneficial Tool for Lead Optimization of Biotherapeutics

In collaboration with Boehringer Ingelheim - Posters

Product Insert Anti 6HIS-Eu / 61HISKLA-61HISKLB

61HISKLA-61HISKLB - Product Insert

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

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Safety Data Sheet Anti 6HIS-Eu / 61HISKLA

61HISKLA - Safety Data Sheet

Safety Data Sheet Anti 6HIS-Eu / 61HISKLB

61HISKLB - Safety Data Sheet

tab6