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HTRF Anti-PT66 mAb Eu-Conjugate, 5,000 Assay Points
HTRF Anti-PT66 mAb Eu-Conjugate, 5,000 Assay Points
MAb PT66-K
MAb PT66-Eu cryptate enables the rapid detection of phosphorylation of peptides or proteins on Tyrosine in a biochemical kinase assay format.
| Feature | Specification |
|---|---|
| Application | 生化学酵素アッセイ |
MAb PT66-Eu cryptate enables the rapid detection of phosphorylation of peptides or proteins on Tyrosine in a biochemical kinase assay format.
Product variants
Unit Size: 5,000 assay points
Part #:
61T66KLA
Unit Size: 20,000 assay points
Part #:
61T66KLB
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Anti-PT66 mAb Eu-Conjugate, 5,000 Assay Points
MAb PT66-K
HTRF Anti-PT66 mAb Eu-Conjugate, 5,000 Assay Points
Product information
Overview
PT66-Eu cryptate is a labeled mouse monoclonal IgG1. It shows to react specifically with phosphorylated tyrosine, both as free amino acid or when conjugated to carriers. No cross-reactivity has been observed with non-phosphorylated tyrosine, phosphothreonine, phosphoserine, AMP or ATP. This allows the construction of a highly specific biochemical Tyrosine kinase assay. Any kind of substrate can be used, from small synthetic peptides to natural substrates, meaning high assay detection versatility. HTRF allows to perform assay at physiological ATP concentrations.
HTRF assays offer many advantages over other technologies:
- Homogeneous add-and-read format
- No wash steps
- Low background
- Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
- Stable signal, providing flexibility in time of readout or size of assays
How it works
Assay principle
HTRF kinase assays typically use a combination of a Europium-cryptate labeled anti-phosphoresidue antibody, SA-XL665 and a biotinylated* substrate. The signal is proportional to the concentration of phospho-residues. *Alternatively, other tags such as 6HIS, GST, c-myc, DNP, or FLAG may be used to label the kinase substrate.
Assay protocol
HTRF kinase assays have two basic phases: 1. Enzymatic step: biotinylated substrate is incubated with the kinase of interest and with the compounds in presence of co-factors. The reaction starts with the addition of ATP. 2. Detection step: The phosphorylated biotinylated substrate is then detected by the addition of Streptavidin-XL665 and MAb PT66-Eu cryptate prepared in a buffer containing EDTA to stop the enzymatic reaction.
Assay validation
Detection at physiological ATP concentration
ATP concentration is an important factor for in vitro kinase assay design, as the sensitivity of ATP competitive inhibitors varies with ATP concentration. As shown in Figure 5, HTRF tolerates the extremely high ATP concentrations (up to mM range) needed for mechanistic studies, e.g. the confirmation of ATP competitive inhibitors. The shift observed for increasing ATP concentrations in the graph confirms the ATP-competitive effect of the compound tested. Assay was performed on a tyrosine kinase revealed by PT66.
Membrane friendly
In addition to fresh and frozen cells, the HTRF Kinase toolbox can be used with membrane preparations. The graph depicts an assay scheme for measuring autophosphorylation of a receptor tyrosine kinase (RTK), using membrane preparations as a source of active kinase. Autophosphorylation of an RTK expressed as a tagged fusion protein is detected using a Eu3+ labeled anti-phospho tyrosine antibody and XL665 labeled anti-tag antibody.
Specifications
| Application |
Biochemical Enzymatic Assay
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Product Group |
Fluorescent Reagent
|
| Shipping Conditions |
Shipped Ambient
|
| Target |
PT66
|
| Target Class |
Kinases
|
| Technology |
TR-FRET
|
| Therapeutic Area |
Cardiovascular
Infectious Diseases
Inflammation
Metabolism/Diabetes
NASH/Fibrosis
Neuroscience
Oncology & Inflammation
Rare Diseases
|
| Unit Size |
5,000 assay points
|
Video gallery
HTRF Anti-PT66 mAb Eu-Conjugate, 5,000 Assay Points
HTRF Anti-PT66 mAb Eu-Conjugate, 5,000 Assay Points
Resources
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Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
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