TransAM® NF?B p52 Chemi

by Active Motif
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48697

Description

TransAM NF?B Kits provide everything needed to study Nuclear Factor ?B (NFkB), including a positive control extract. The TransAM NF?B Chemi Kits couple the TransAM method to highly sensitive chemiluminescent detection. This format offers improved sensitivity and a greater dynamic range. Kits are available with antibodies specific for the activated form of p50, p52 and p65. Recombinant p50 and Recombinant p65 proteins are available separately to generate an optional protein standard curve in the TransAM NF?B Transcription Factor ELISA kits. The TransAM® Chemi tab below provides complete method details; the NFkB Info tab gives an overview of the NF?B transcription factor as well as kit data; the kit manual can be downloaded under the Documents tab.

 
TransAM NFkB p50 Chemi sensitivity 
 
TransAM NF?B p50 Chemi sensitivity.

Nuclear extracts from Jurkat cells and Jurkat cells stimulated with TPA and calcium ionophore were assayed from 0.039 to 2.5 µg/well for NF?B p50 activation using the TransAM NF?B p50 Chemi Kit.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NF?B-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).

 
Name Format Cat No. Price  
TransAM® NF?B p50 Chemi 1 x 96 rxns 41097 $740 Buy Now
TransAM® NF?B p50 Chemi 5 x 96 rxns 41597 $3,025 Buy Now
TransAM® NF?B p52 Chemi 1 x 96 rxns 48197 $740 Buy Now
TransAM® NF?B p52 Chemi 5 x 96 rxns 48697 $3,025 Buy Now
TransAM® NF?B p65 Chemi 1 x 96 rxns 40097 $740 Buy Now
TransAM® NF?B p65 Chemi 5 x 96 rxns 40597 $3,025 Buy Now

The TransAM® Chemi transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

Why use TransAM® Chemi transcription factor ELISAs?

  • Up to 100-fold more sensitive than gelshift assays
  • Eliminates the use of radioactivity and the need to run gels
  • Results in less than five hours
  • Luminescent readout enables easy, quantitative analysis with a luminometer or CCD camera system
  • 96-stripwell format enables both high and low throughput

How TransAM® Chemi transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Chemiluminescent Reagent to provide an easily quantified, sensitive chemiluminescent readout (Figure 1).

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Figure 1: Flow chart of the TransAM Chemi process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NF?B-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).

Contents & Storage

One or five NF?B 96-well assay plate(s) with plate sealer(s), NF?B p50, p52 or p65 primary antibody, HRP-conjugated secondary antibody, NF?B wild-type and mutated oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Herring Sperm DNA, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, and Chemiluminescent Substrate Solutions. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.

NOTE: the p50 and p52 antibodies can be used with human and mouse extracts, while the p65 antibody can be used with human, mouse and rat extracts.

 

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NF?B-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).

TransAM NF?B Chemi Kits: Catalog Number 40097

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