HTRF mAb Anti-GST Eu-Conjugate, 5,000 Assay Points

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HTRF mAb Anti-GST Eu-Conjugate, 5,000 Assay Points

Eu cryptate-labeled anti-GST antibody for capturing GST-tagged proteins in protein/protein interaction assays.

For research use only. Not for use in diagnostic procedures.

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Feature	Specification
Application	Protein-Protein Interaction

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Eu cryptate-labeled anti-GST antibody for capturing GST-tagged proteins in protein/protein interaction assays.

For research use only. Not for use in diagnostic procedures.

View product information

Product Variants

Unit Size: 5,000 Assay Points

Part #:

61GSTKLA

List Price

USD 609.47

Unit Size: 20,000 Assay Points

Part #:

61GSTKLB

List Price

USD 1,911.00

Product information

Overview

MAb Anti GST-Eu cryptate is an IgG2a raised against Glutathione S-transferase labeled with Eu. It has been shown to react with GST-tagged fusion proteins from a large number of expressing vectors.

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.

Specifications

Other Specifications

Application	Protein-Protein Interaction
Brand	HTRF
Detection Modality	HTRF
Product Group	Fluorescent Reagent
Shipping Conditions	Shipped Ambient
Target Class	Binding Assay
Technology	TR-FRET
Therapeutic Area	Cardiovascular Infectious Diseases Inflammation Metabolism/Diabetes NASH/Fibrosis Neuroscience Oncology & Inflammation Rare Diseases
Unit Size	5,000 Assay Points

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Citations

View product citations for 61GSTKLA on CiteAb

How it works

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 GST-Eu cryptate binds to the GST 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 protocol

The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a GST-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti GST-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.

Assay details

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 Revvity, 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.

Assay validation

p53/HDM2 binding assay

p53, a tumor suppressor protein activated in response to DNA damage, is regulated by the binding of HDM2 which induces ubiquitin-medicated degradation of p53. Our HTRF assay was developed to monitor p53/HDM2 binding, to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p53 homologue, p73, for HDM2. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody. Kane et al. - Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence. Anal Biochem. 2000;278: 29-38.