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Gene Regulation

Luminex® Transcription Factor Assays

study analyte-specific DNA binding activity in multiplex

Active Motif is a provider of Transcription Factor Multiplex Assays for use with the Luminex® 200™ or MAGPIX® instruments. The Transcription Factor Multiplex Kits are novel bead-based assays designed to study analyte-specific DNA binding activity of proteins belonging to the same transcription factor family. Antibodies specific to each transcription factor protein are conjugated to a unique Luminex bead to enable comparative analysis of DNA binding between proteins in the same family. Kits are available for the study of AP-1 and NFκB transcription factor families.

Each Multiplex Kit contains a DNA probe of the transcription factor consensus binding sequence, competitor oligonucleotides, a positive control lysate, 96-well assay plate and the necessary buffers and detecting reagents to perform the assay. Antibody-conjugated beads must be purchased separately to complete the assay. The individual bead packaging allows researchers to customize their assays for singleplex or multiplex analysis simply by selecting antibody-conjugated beads for their protein(s) of interest. To learn more about how the Transcription Factor Multiplex Assays work or to read about the Luminex* technology, click on the Method tab below.

Note: A complete assay requires the purchase of both the Transcription Factor Multiplex Kit containing the buffers and the antibody-conjugated bead(s) of interest.

*Luminex is a registered trademark of the Luminex Corporation. Users of the assay products are agreeing to the Luminex terms and conditions as stated in the product manual.

 
Name Format Cat No. Price  
Transcription Factor Multiplex Kit - AP-1 96 rxns 33100 $425 Buy Now
c-Fos-conjugated beads 48 rxns 33101 $180 Buy Now
FosB-conjugated beads 48 rxns 33102 $180 Buy Now
JunB-conjugated beads 48 rxns 33104 $180 Buy Now
JunD-conjugated beads 48 rxns 33105 $180 Buy Now
Transcription Factor Multiplex Kit - NFκB 96 rxns 33110 $425 Buy Now
NFκB p65-conjugated Beads 48 rxns 33113 $180 Buy Now
NFκB p50-conjugated Beads 48 rxns 33114 $180 Buy Now
NFκB p52-conjugated beads 48 rxns 33112 $180 Buy Now
RelB-conjugated beads 48 rxns 33111 $180 Buy Now

How Do Transcription Factor Multiplex Kits Work?

The Transcription Factor Multiplex Assays work as pull-down reactions where cell lysates are combined with a biotinylated double-stranded consensus binding sequence for the transcription factor family of interest. Fluorescently labeled magnetic beads, which have been conjugated to antibodies for the individual transcription factor family members, are used to capture the transcription factor-bound DNA. Streptavidin-phycoerythrin is used to bind the biotinylated oligo and determine the magnitude of signal (see Figure 1).

Since each antibody-conjugated bead contains a different fluorescent label, antibodies for multiple family members can be added to the same sample for multiplexing. The readout from the streptavidin-phycoerythrin is proportional to the amount of transcription factor binding. This allows for comparative analysis of DNA binding activity within the same transcription factor family. Active Motif offers buffer kits and antibody-conjugated magnetic beads to create your own custom multiplex or singleplex assays for the study of AP-1 and NFκB transcription factor families.

Schematic illustrating the principles of the Luminex Transcription Factor Multiplex Assay
Figure 1: Transcription Factor Multiplex Kits are bead-based assays to measure transcription factor-DNA binding activity.

 

The Luminex Principle

The Luminex technology is a bead-based assay system using tiny, 6.4 micron superparamagnetic beads. Each bead contains its own distinct dye ratio which will generate a unique fluorescence pattern to enable individual bead identity. Active Motif provides the beads conjugated to transcription factor antibodies which can be used individually or in combination to capture the protein(s) of interest from the sample. Once the beads are identified by their distinct fluorescence within the Luminex instrument, the identity of the captured protein is also known. To assess DNA-binding activity, a biotinylated DNA oligonucleotide is also included in the assay. Streptavidin-phycoerythrin will bind to the biotinylated DNA and produce a fluorescence signal proportional to the DNA binding activity.

If using the Luminex® 200™ instrument, the beads are individually passed through a small shaft where a light source excites the internal dye that is used to identify each bead. Based on the dye ratios, each bead will exhibit a unique fluorescence that can be identified with a unique bead number. A second wavelength is used to determine the magnitude of the streptavidin-phycoerythrin signal (see Figure 2). A minimum of 100 beads are read for each well of the assay. If using the MAGPIX® instrument, beads are collected as a monolayer on the sample chamber. LEDs are used to illuminate the chamber and CCD imaging technology capture a series of images. The software analyzes the images to determine the bead identity and phycoerythrin signal. The Luminex xPONENT® software program will provide a real-time readout of signal measured as median fluorescent intensity (MFI).

Luminex 200 instrument reading
Figure 2: The Luminex® 200™ instrument uses two distinct wavelengths to analyze individual beads as they pass through the machine for bead identity and phycoerythrin signal.

Transcription Factor AP-1

AP-1 is a transcription factor family consisting of a mixture of homodimeric and heterodimeric protein complexes derived from the Fos and Jun families. The AP-1 proteins play a role in the expression of genes involved in regulation of differentiation, proliferation and apoptosis. AP-1 expression is induced by multiple stimuli, including the presence of serum, growth factors, phorbol esters and oncogenes. Phosphorylation of AP-1 family members by kinases is required for transactivation activity. Once the AP-1 family members have translocated into the nucleus they are able to bind DNA. Active Motif offer antibody-conjugated beads for each of the following AP-1 family members: c-Fos, FosB, Fra-1, JunB and JunD.

Transcription Factor AP-1 Singleplex and Multiplex Assays
Figure 1: DNA binding activity of AP-1 transcription factor family members in either singleplex or multiplex analysis.

The Transcription Factor Multiplex Kit – AP-1 was used to study the DNA binding activity of various members of the AP-1 transcription factor family. The top images show singleplex data for FosB and JunB assays run individually. The positive control lysate, K-562 nuclear extract treated with TPA, is tested in the absence or presence of the provided competitor oligos. The wild-type oligo shares the same consensus sequence as the biotinylated consensus oligonucleotide. Therefore, it competes for binding with the transcription factor, inhibiting the ability of the transcription factor to bind the biotinylated consensus oligonucleotide. The mutant oligonucleotide has a modified consensus sequence that is not recognized by the transcription factor and therefore does not compete for transcription factor binding. The use of the wild-type and mutant oligos within the assay confirms the specificity of the assay. The bottom image shows a multiplex assay in which multiple transcription factor antibody-conjugated beads were combined into a single well of the assay to study the transcription factor activity of various AP-1 family members within the same sample. A comparison of the top and bottom images shows that multiplexing does not have any negative effects on the assay results.

Transcription Factor NFκB

NFκB is a widely studied transcription factor due to its implications in the regulation of genes that control inflammation, cell proliferation and cell survival. The NFκB family is comprised of a mixture of homodimeric and heterodimeric subunits that are members of the structurally related Rel family of transcription factors. NFκB p65 (RelA), RelB, and c-Rel contain a transactivation domain that is required for translocation of active NFκB complexes into the nucleus where they are able to bind DNA. The p50 and p52 subunits do not contain the transactivation domain and must for heterodimers. Active Motif currently offers antibody-conjugated beads for the NFκB p65, NFκB p50, NFκB p52 and RelB subunits.

Luminex NFkB Multiplex
Figure 1: 4-plex analysis of NFκB family members.

The Transcription Factor Multiplex Kit – NFκB was used to study the DNA binding activity of various members of the NFκB transcription factor family within the same sample. The antibody-conjugated beads for the different NFκB family members were multiplexed together within the same assay well. The positive control lysate, Raji nuclear extract, is tested in the absence or presence of the provided competitor oligos. The wild-type oligo shares the same consensus sequence as the biotinylated consensus oligonucleotide. Therefore, it competes for binding with the transcription factor, inhibiting the ability of the transcription factor to bind the biotinylated consensus oligonucleotide. The mutant oligonucleotide has a modified consensus sequence that is not recognized by the transcription factor and therefore does not compete for transcription factor binding. The use of the wild-type and mutant oligos within the assay confirms the specificity of the assay.

 

Luminex NFkB Multiplex
Figure 2: Singleplex NFκB p50 analysis and quantitation using a Recombinant NFκB p50 protein.

The Transcription Factor Multiplex Kit – NFκB was used to analyze NFκB p50 DNA-binding activity from Raji nuclear extract. The Raji extract was tested in the presence and absence of the competitor oligonucleotides to show specificity (top image). A standard curve was generated in a separate assay well using Active Motif's Recombinant NFκB p50 protein (Catalog No. 31101) and the NFκB p50-conjugated beads (bottom image) in order to quantify the amount of NFκB p50 binding within the Raji lysate.

Contents & Storage

The Transcription Factor Multiplex Kits contain the necessary buffers and reagents to perform the assay, but they do not include any antibody-conjugated beads. Beads must be purchased separately.

Transcription Factor Multiplex Kit – AP-1 or NFκB

  • Biotinylated AP-1/NFκB Consensus Probe; Store at -20°C
  • Wild-type AP-1/NFκB oligonucleotide; Store at -20°C
  • Mutant AP-1/NFκB oligonucleotide; Store at -20°C
  • 1 M DTT; Store at -20°C
  • Poly [d(I-C)]; Store at -20°C
  • Protease Inhibitor Cocktail; Store at -20°C
  • Lysis Buffer AM2; Store at 4°C
  • Binding Buffer AM1; Store at 4°C
  • 10X Antibody Binding Buffer AM2; Store at 4°C
  • Streptavidin-PE; Store at 4°C
  • 96-well assay plate; Store at RT
  • Plate sealer; Store at RT
  • Positive control nuclear extract; Store at -80°C

Antibody-conjugated Beads

  • Transcription factor-specific antibody-conjugated beads; Store at 4°C