US
Search Results
Revvity Sites Globally
Select your location.
*e-commerce not available for this region.
HTRF MAb Anti GST-Eu cryptate, 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.
Product Variants
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
| Assay Points |
5000
|
|---|---|
| Assay Target Type |
Fluorescent reagent
|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Therapeutic Area |
Cardiovascular
Infectious Diseases
Inflammation
Metabolism/Diabetes
NASH/Fibrosis
Neuroscience
Oncology & Inflammation
Rare Diseases
|
| Unit Size |
5,000 Assay Points
|
Video gallery
Citations
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.
Resources
Application Note
Download your application note on nuclear receptor ligand identification with HTRF
Dive deeper into NASH investigation.
Nuclear Receptors (NRs) regulate gene transcription by DNA binding. Interaction of NRs with an...
Application Note
Enhance PROTAC Drug Discovery with AlphaLISA and HTRF Kits
A Bruton Tyrosine Kinase (BTK) case study
PROteolysis Targeting Chimeras (PROTACs) are one of the latest trending tools used in...
Application Note
HTRF addresses large protein-protein interaction complexes
Challenge large complexes with HTRF assays
This note describes how to investigate small- to large protein-protein interaction...
Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
Application Note
Improve PROTAC Targeting Kinase Discovery with HTRF Binding Kits
HTRF assays to assess PROTAC® compounds
Kinases constitute about 2% of all human genes and are essential proteins involved in...
eBook
Protein Degradation - A path to new Therapeutics eBook
Get up to speed with the latest developments on PROTAC® design and verification.
Harnessing the power of the cellular protein and...
SDS, COAs, Manuals and more
Are you looking for technical documents for this product. We have housed them in a dedicated section., click on the links below to explore.
How can we help you?
We are here to answer your questions.
