NFkB Family EZ-TFA Transcription Factor Assay Chemiluminescent Kit

NFkB Family EZ-TFA Transcription Factor Assay Chemiluminescent Kit

Upstate (Millipore)

2 x 96 assays

The Upstate® Non-Radioactive NFκB Family EZ-TFA provides a fast, sensitive method to detect specific transcription factor DNA binding activity in nuclear extracts. This assay combines the principle of the electrophoretic mobility shift assay (EMSA) with the 96-well based enzyme-linked immunosorbent assay (ELISA), enabling manual or high-throughput sample analysis with greater sensitivity than conventional EMSA assays. The entire assay takes less than 4 hours to complete with minimal hands-on time. The versatile set up allows for a flexible assay design and because the binding reaction occurs in solution, various parameters can be optimized such as probe titration, competitive oligonucleotide concentration, or treatment conditions.


Introduction:


The NFκB (Nuclear Factor κ B) transcription factor family is involved in the expression and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response, and activation of various viral promoters including human immunodeficiency virus long terminal repeats (DiDonato 1995; Huang 2001). NFκB represents a group of structurally related and evolutionarily conserved proteins related to the proto-oncogene c-Rel with five members in mammals that include Rel (cRel), RelA (p65), RelB, NFκB1 (p50 and its precursor p105), and NFκB2 (p52 and its precursor p100)(DiDonato 1995; Huang 2001). NFκB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFκB dimers are activators of transcription, the p50/50 and p52/52 homodimers can repress the transcription of their target genes (Huang 2001; Ghosh 2002). The p50/p65 heterodimer of NFκB is the most abundant in cells(Ghosh 2002).

A critical component in NFκB regulation is the IκB Kinase (IKK) complex (Ghosh 2002). In a majority of unstimulated cells, the NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IkaapaB proteins (Baeuerle 1988; Miyamoto 1994). The NFκB complex is activated upon stimulation by multiple inducers including viruses or cytokines. The classical p50/p65 (RelA) NFκB heterodimer complex is activated by ligands such as TNFα, IL-1, or PMA. This signal transduction event causes the rapid phosphorylation of IκBalpha and it's subsequent degradation. This results in the release of the NFκB p105/p65 heterodimer complex. The p105 subunit is then cleaved into its active p50 form. This cleavage exposes the NLS sequence on the p50 subunit. The p50/p65 heteroduplex then translocates to the nucleus where it activates target gene transcription. NFκB induces the transcription of its own inhibitor, IκBalpha, causing an autoregulatory mechanism of NFκB activity and generating the inactive form of NFκB. The newly formed nuclear NFκB-IκBalpha complexes are then exported out to the cytoplasm, thereby re-establishing the cytoplasmic pool of inactive NFκB complexes primed for another round of activation to take place (Huang 2001). The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes (Ghosh 2002). In an alternative NFκB pathway involving the p52/RelB heterodimers, activation is dependent upon LTα/β, CD40L, and Blys/BAFF, and not the activators that induce the Classical pathway (Karin 2004). This pathway has been found to be important in secondary lymphoid organ development, maturation of B cells, and adaptive humoral immunity (Karin 2004).



Test Principle:

The Non-Radioactive NFκB Family Transcription Factor Assay kit is provided in a 96-well format. During the assay, the NFκB Capture Probe, a double stranded biotinylated oligonucleotide containing the flanked DNA binding consensus sequence for NFκB (5'-GGGACTTTCC-3'), is mixed with cellular (nuclear) extract in the Transcription Factor Assay Buffer provided. When incubated together, the active form of NFκB contained in the nuclear extract binds to its consensus sequence. The extract/probe/buffer mixture is then directly transferred to the streptavidin-coated plate. The active NFκB protein is immobilized on the biotinylated double stranded oligonucleotide capture probe bound to the streptavidin plate well, and any inactive, unbound material is washed away. The bound NFκB transcription factor subunits are detected with specific primary antibodies directed against each individual specific target. An HRP-conjugated secondary antibody is then used for detection. This provides sensitive chemiluminescent detection that can be read in a microplate luminometer or by a CCD camera-coupled imaging system. Included in the kits are positive cell extract, a non-specific double stranded oligonucleotide, and a specific competitor double stranded oligonucleotide.

For Research Use Only; Not for use in diagnostic procedures

Use of this assay in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and requires a license from Ariad Pharmaceuticals (Cambridge, MA, USA).

The transcription factor NFκB (Nuclear Factor kappa B) is involved in the expression and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response, and activation of various viral promoters including human immunodeficiency virus long terminal repeat. NFκB represents a group of structurally related and evolutionarily conserved proteins related to the proto-oncogene c-Rel with five members in mammals that include Rel (cRel), RelA (p65), RelB, NFκB1 (p50 and its precursor p105), and NFκB2 (p52 and its precursor p100). NFκB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFκB dimers are activators of transcription, the p50/50 and p52/52 homodimers can repress the transcription of their target genes. The p50/p65 heterodimer of NFκB is the most abundant in cells3.
A critical component in NFκB regulation is the IκB Kinase (IKK) complex. In a majority of unstimulated cells, the NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IκB proteins. Upon stimulation by multiple inducers including viruses or cytokines, such as TNFα, IL-1, or PMA, IκBκ is rapidly phosphorylated and degraded, resulting in the release of the NFκB complex, most commonly the p105/p65 heterodimer. The p105 subunit is cleaved into its active p50 form. This cleavage exposes the NLS sequence on the p50 subunit. The p50/p65 heteroduplex then translocates to the nucleus where it activates gene transcription. NFκB induces the transcription of its own inhibitor, IκBα, causing an autoregulatory mechanism of NFκB activity and generating the inactive form of NFκB. The newly formed nuclear NFκB-IκBα complexes are then exported out to the cytoplasm, thereby reestablishing the cytoplasmic pool of inactive NFκB complexes primed for another round of activation to take place. The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes.

• Activity Assay
• Affinity Binding Assay

• Human
• Mouse
• Rat

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA, or RELB (MIM 604758) to form the NFKB complex. The p50 (NFKB1)/p65 (RELA) heterodimer is the most abundant form of NFKB. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA, MIM 164008 or NFKBIB, MIM 604495), which inactivate NFKB by trapping it in the cytoplasm. Phosphorylation of serine residues on the I-kappa-B proteins by kinases (IKBKA, MIM 600664, or IKBKB, MIM 603258) marks them for destruction via the ubiquitination pathway, thereby allowing activation of the NFKB complex. Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine).[supplied by OMIM]

FUNCTION: SwissProt: Q04864 # Proto-oncogene that may play a role in differentiation and lymphopoiesis. May function as a transcriptional transactivator.
SIZE: 619 amino acids; 68520 Da
SUBUNIT: Homodimer (By similarity). Interacts with NKIRAS1.
SUBCELLULAR LOCATION: Nucleus (By similarity).
SIMILARITY: SwissProt: Q04864 ## Contains 1 RHD (Rel-like) domain.

The reagents in this kit have been optimized to reduce background signal while maintaining a high signal to noise ratio. Quality control testing is performed using the enclosed kit components while following the protocol described herein.

• EZ-TFA (Universal Transcription Factor Assay, Chemiluminescent) Catalog #70-601. One universal kit containing 125 mL TFA Buffer, two streptavidin-coated stripwell plate, 24 grams of blocking reagent, 250 μl 10X annealing buffer, two 4 mL chemiluminescent detection reagent, two 8 mL chemiluminescent reaction buffer, 60 μl Gt X Rb HRP secondary antibody, 60 μl Gt X Ms HRP secondary antibody. A detailed manual is also included with the 70-601 Universal TFA component. The procedures described in the manual should be consulted as well as the specific product information in this 70-660 product specific certificate of analysis.
• NFκB p65 Primary Antibody (Part No. 90274): One vial containing 15 μL Rabbit anti-NFκB p65.
• NFκB p52 Primary Antibody (Part No. 2003489): One vial containing 15 μL of anti-NFκB p52.
• NFκB p50 Primary Antibody (Part No. 90275): One vial containing 15 μL of anti-NFκB p50.
• RelB Primary Antibody (Part No. 2003492): One vial containing 15 μL of anti-RelB.
• c-Rel Primary Antibody (Part No. 2003491): One vial containing 15 μL of anti-c-Rel.
• NFκB Specific Competitor Probe (Part No. 2003485): One vial containing 40 μL (10 pmol/μL) specific competitor oligonucleotide probe. The NFκB Competitor Probe has the same consensus sequence (5'-GGGACTTTCC-3') as the NFκB Capture Probe but is not biotinylated and will not bind to the plate. It is provided as a specific competitor for transcription factor binding and can be used to demonstrate specificity. This control does not need to be run with each assay performed.
• NFκB Capture Probe (Part No. 2003484): One vial containing 440 μL biotinylated double stranded oligonucleotide probe (1 pmol/μL) containing the wild-type consensus sequence (5'-GGGACTTTCC-3') for NFκB.
• TFA Negative Control Probe (Part No. 2003486): One vial containing 40 μL biotinylated double stranded oligonucleotide probe (2 pmol/μL). Provided as an internal negative control, this non-specific competitor oligonucleotide does not contain the NFκB consensus sequence.
• HeLa Whole Cell Extract, TNFα Treated (Part No. 90350): One vial containing 25 μL TNFα stimulated HeLa whole cell extract. This extract is provided as a positive control and is not intended for extract-to-extract, nor plate-to-plate comparisons. When using this control, you will notice a decrease in signal with repeated freeze/thaw cycles. This component must be stored at -80°C.
• Raji Nuclear Extract (Part No. 2003495): One vial containing 25 μL Raji nuclear extract. This extract is provided as a positive control and is not intended for extract-to-extract, nor plate-to-plate comparisons. When using this control, you will notice a decrease in signal with repeated freeze/thaw cycles. This component must be stored at -80°C.

Upstate

• P19838
• Q00653
• Q04206
• Q04864
• Q01201

The Transcription Factor Assay is a triple temperature storage assay kit. Store kit components at the temperatures indicated on the labels and insert until their expiration date.

• Store the NFkappaB Capture Probes, Competitor Oligonucleotides, Negative Control Probes, and all antibodies at -20°C.

• Store the Transcription Factor Assay (TFA) Buffer at -20°C. Avoid freeze/thawing of the TFA Buffer. Buffer can be thawed in warm water prior to use. Buffer can be aliquotted and frozen.

• Store HeLa and Raji Extracts at -80°C. Avoid repeated freeze/thawing. Always keep on ice when using. Stable at -80°C for 6 months.

• Store the TFA 96-well plates and the TMB/E and Stop solutions at 4°C.

2 x 96 wells per plate

The transcription factor NFκB (Nuclear Factor kappa B) is involved in the expression and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response. NFκB represents a group of 5 structurally related and evolutionarily conserved proteins in mammals that include Rel (cRel), RelA (p65), RelB, NFκB1 (p50 and its precursor p105), and NFκB2 (p52 and its precursor p100). NFκB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes, though the p50/p65 heterodimer of NFκB appears to be the most abundant in cells. A critical component in NFκB regulation is the IκB Kinase (IKK) complex. NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IκB proteins. Upon stimulation by multiple inducers including viruses or cytokines, IκB is rapidly phosphorylated and degraded, resulting in the release of the NFκB complex. The p105 subunit is cleaved into its active p50 form. This cleavage exposes the NLS sequence on the p50 subunit. The p50/p65 heteroduplex then translocates to the nucleus where it activates gene transcription. The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins and inducible effector enzymes.

• NFKB1
• NF-kappa-B
• NFKB-p105
• EBP-1
• KBF1
• DKFZp686C01211
• MGC54151
• NFKB-p50

NFkB Family EZ-TFA Transcription Factor Assay Chemiluminescent Kit

NM_003998.2

NFκB Family (p50, p52, p65 (RelA), c-Rel, RelB )

1. Nuclear Extraction Kit (Catalog Number 2900) or components
2. Pipettor (both single and multi-channel)
3. Pipette tips
4. Distilled water
5. Rotator or Plate shaker (optional)
6. Plate cover (optional)
7. Microplate luminometer or CCD camera-coupled imaging system