Human CCL4 (MIP1 beta) kit
No-wash kit to quantify released Human CCL4 (MIP1 beta)
Phospho-IKK-beta (Ser177/181) cellular kit HTRF®
The phospho-IKK-beta (Ser177/181) assay enables the cell-based detection of Ser177/181 phosphorylation of activated IKK-beta directly in whole cells.
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Ready-to-use
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Faster and more convenient than ELISA
Overview
The Phospho-IKK-beta (Ser177/181) assay enables the detection of Ser177/181 phosphorylation of activated 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.
Benefits
- SPECIFICITY
- PRECISION
Phospho-IKKß (Ser177/181) assay principle
The phospho-IKK-beta (Ser177/181) assay measures IKK beta when phosphorylated at Ser177/181. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The phospho-IKK-beta (Ser177/181) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.
Phospho-IKKß (Ser177/181) 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 Phospho-IKK beta (Ser177/181) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-IKKß (Ser177/181) 1-plate assay protocol
Detection of Phosphorylated IKK-beta (Ser177/181) 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.
HTRF phospho-IKKß assay compared to Western Blot
HeLa cells were grown in a T175 flask 37°C, 5% Co2, 2 days. Stimulation was done with IL1b 0.5nM for 15min. After elimination of cell culture medium, 3 mL of supplemented lysis buffer was added and incubated for 45min. Soluble supernatants were collected after 10min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. HTRF assay shows better sensitivity than Western Blot: 12,000 cells for HTRF compared to 46,000 cells for Western Blot.
TNFa dose-response on HeLa cells
Two concentrations of HeLa cells (100 and 200K cells/well) were incubated for 15 minutes at 37°C with various concentrations of TNF alpha. After a 30 minutes lysis incubation time, phosphorylated IKK-beta was measured using the two-plate assay protocol.
IL1ß dose-response on HeLa cells
Two concentrations of HeLa cells (100 and 200K cells/well) were incubated for 15 minutes at 37°C with various concentrations of IL1 beta. After a 30 minutes lysis incubation time, phosphorylated IKK-beta was measured using the two-plate assay protocol.
Detection of phospho and total IKK-beta on HeLa cells
Different cell densities (200K and 100K) of HeLa cells were plated under 100µL in 96-well plate and incubated overnight. Media was aspirated and 50µL of different concentrations of IL-1beta was added during 15 minutes. After incubation, media was aspirated and cells were lysed with 50µL 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 IKKbeta (Ser177/181) or total IKKbeta detection reagents were added. The HTRF signal was recorded after a 2 hour incubation at room temperature.
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.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Investigating kinase activity in a cellular context
HTRF cellular assays - Scientific Presentations
Open R&D: Sanofi Access Platform
In collaboration with Sanofi - Scientific Presentations
Translating academic research into early stage drug discovery projects
In collaboration with European ScreeningPort - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF and Alpha solutions for NASH - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
HTRF assays for Oncology and Inflammation
Signaling in the Immune System - Brochures
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Characterize STING agonists with HTRF human IFNb
Data about HTRF IFNb correlation with gene reporter assay and ELISA - Application Notes
An innate and adaptive immunity recap
Insight into the diversity of cells & signaling pathways - Guides
Assays for neurosciences research
Advance your research on neurodegenerative diseases - Flyers
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Manual IKK b P-S177/181 Kit / 64KKBPEG-64KKBPEH
64KKBPEG-64KKBPEH - Product Insert
Certificate of Analysis IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG Batch 06B - Quality Control Report
Safety Data Sheet (DEU) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (ELL) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (ITA) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (SPA) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) IKK b P-S177/181 Kit / 64KKBPEG
64KKBPEG - Safety Data Sheet
Safety Data Sheet (ELL) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (ITA) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (SPA) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (DEU) IKK b P-S177/181 Kit / 64KKBPEH
64KKBPEH - Safety Data Sheet
Safety Data Sheet (DEU) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
Safety Data Sheet (ELL) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
Safety Data Sheet (ITA) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
Safety Data Sheet (SPA) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
Safety Data Sheet (ENG-US) IKK b P-S177/181 Kit / 64KKBPEY
64KKBPEY - Safety Data Sheet
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