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HTRF Human Total IκKβ Detection Kit, 500 Assay Points
HTRF Human Total IκKβ Detection Kit, 500 Assay Points
generic HTRF total primary image
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Sample Volume | 16 µL |
Product variants
Unit Size: 500 assay points
Part #:
63ADK097PEG
Unit Size: 10,000 assay points
Part #:
63ADK097PEH
Unit Size: 50,000 assay points
Part #:
63ADK097PEY
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 Human Total IκKβ Detection Kit, 500 Assay Points
generic HTRF total primary image
HTRF Human Total IκKβ Detection Kit, 500 Assay Points
Product information
Overview
The total-IKK-beta assay enables the detection of total IKK-beta directly in whole cells. Using a streamlined protocol, amenable to low-volume formats, this kit can be used from basic research to High Throughput drug screening.
How it works
Total-IKKß assay principle
The Total IKK-beta assay quantifies the expression level of IKK-beta in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total IKK-beta assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of IKKß in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format.
Total-IKKß 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total IKK-beta HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-IKKß cellular 1-plate assay protocol
Detection of total IKK-beta cellular with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Assay validation
Detection of phospho and total IKK-beta on HeLa cells
Different cell densities (200K and 100K) of HeLa cells were plated under 100 µL in 96-well plate and incubated overnight. Media was aspirated and 50 µL of different concentrations of IL-1 beta was added during 15 minutes. After incubation, media was aspirated and cells were lysed with 50 µL of lysis buffer 1X for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho IKK-beta (Ser177/181) or total IKK-beta detection reagents were added. The HTRF signal was recorded after a 2 hour incubation at room temperature.
Simplified pathway
IKKß Simplified Pathway
Activation of the NF-?B is initiated by the signal-induced degradation of I?B proteins. This occurs primarily via activation of a kinase called the I?B kinase (IKK). IKK is composed of a heterotrimer of 3 subunits, IKK-alpha and IKK-beta (the two catalytic subunits) and IKK-gamma/NEMO (a regulatory component). Activated IKK-beta phosphorylates a protein called the inhibitor of NF-?B, I?B (I?Ba), which binds NF-?B to inhibit its function. Phosphorylated I?B is degraded via the ubiquitination pathway, freeing NF-?B and allowing its entry into the nucleus of the cell, where it activates various genes involved in inflammation and other immune responses. IKK-beta plays a significant role in brain cells following a stroke.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Lysis Buffer Compatibility |
Lysis Buffer 1
Lysis Buffer 4
Lysis Buffer 5
|
| Molecular Modification |
Total
|
| Product Group |
Kit
|
| Sample Volume |
16 µL
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
TR-FRET
|
| Therapeutic Area |
Infectious Diseases
Metabolism/Diabetes
NASH/Fibrosis
Neuroscience
Oncology & Inflammation
|
| Unit Size |
500 assay points
|
Video gallery
HTRF Human Total IκKβ Detection Kit, 500 Assay Points
HTRF Human Total IκKβ Detection Kit, 500 Assay Points
Resources
Are you looking for resources, click on the resource type to explore further.
Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
Guide
Protein degradation awareness in a single guide
An in-depth review of molecular and cellular pathways
The maintenance of proteostasis, the biological mechanisms that control the...
Application Note
TCR phospho signaling investigation with HTRF
Study your pathway of interest in PBMC and T cells
Combine models with a technique that maximizes each one’s relevance: this note...
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