货号：70-560

品牌：Merck Millipore

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目录价：￥12648.00

市场价格：￥10750.80

会员价格：￥10118.40

金山科研平台，产品价格货期咨询微信：jinshanbio Description: NFκB Family EZ-TFA Transcription Factor Assay Colorimetric Kit View All» Trade Name: Upstate (Millipore) View All» Qty/Pk: 2 x 96 assays View All» Product Overview: The Upstate® Non-Radioactive NFκB Family Transcription Factor Assay 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 colorimetric detection that can be read in a spectrophotometric plate reader. 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). View All» Key Applications: Activity Assay View All» Species Reactivity:

• Human

• Mouse

• Rat
  View All» Usage Statement: 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. View All» Entrez Gene Summary: 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] View All» UniProt Summary: 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. View All» Brand Family: Upstate View All» Storage Conditions: 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. View All» Replaces: SGT560 View All» Gene Symbol:
  • NFKB1

  • NF-kappa-B

  • NFKB-p105

  • EBP-1

  • KBF1

  • DKFZp686C01211

  • MGC54151

  • NFKB-p50
    View All» Product Name: NFκB Family EZ-TFA Transcription Factor Assay Colorimetric Kit View All» Alternate Names: NFκB Family (p50, p52, p65 (RelA), c-Rel, RelB ) View All» Materials Required but Not Delivered: 1. Nuclear Extraction Kit (Catalog No. 2900) or suitable extraction reagents 2. Pipettor (both single and multi-channel) 3. Pipette tips 4. Distilled water 5. Microplate reader (wavelength 450nm/650nm (650 optional)) 6. Rotator or Plate shaker (optional) 7. Plate cover (optional) View All» Quality Assurance: 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. View All» Components:
    • Transcription Factor Assay Plate (Part No. 90268): Two 96-well Streptavidin coated plates sealed in a foil pouch with desiccant.

    • TFA Buffer, 5x (Part No. 2003487): One bottle containing 125 mL of 5x Transcription Factor Assay Buffer containing sonicated salmon sperm DNA to block non-specific DNA binding activity.

    • Blocking Reagent (Part No. 2003488): One vial containing 24 grams of Blocking Reagent.

    • p50 Primary Antibody (Part No. 90275): One vial containing 15 μL of anti-NFκB p50. Use at 1:1000 dilution in Enhanced Transcription Factor Assay Buffer (see Assay Protocol).

    • p52 Primary Antibody (Part No. 2003489): One vial containing 15 μL of anti-NFκB p52. Use at 1:1000 dilution in Enhanced Transcription Factor Assay Buffer (see Assay Protocol).

    • p65 Primary Antibody (Part No. 90274): One vial containing 15 µL Rabbit anti-NFκB p65. Use at 1:1000 dilution in Enhanced Transcription Factor Assay Buffer (see Assay Protocol).

    • RelB Primary Antibody (Part No. 2003492): One vial containing 15 μL of anti-RelB. Use at 1:500 dilution in Enhanced Transcription Factor Assay Buffer (see Assay Protocol).

    • c-Rel Primary Antibody (Part No. 2003491): One

    • vial containing 15 μL of anti-c-Rel. Use at 1:250

    • dilution in Enhanced Transcription Factor Assay

    • Buffer

    • Gt X Rb, HRP Secondary Antibody (Part No. 2003494): One vial containing 60 μL Goat anti-Rabbit, HRP conjugated secondary detection antibody. Use at 1:500 dilution in Enhanced Transcription Factor Assay Buffer.

    • 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.

    • 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.

    • 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.

    • Substrate (TMB/E) (Part No. 90348): Two 12 mL bottles (Ready to use).

    • (Caution: Caustic material. Eye, hand, face, and clothing protection should be worn when handling this material.)

    • Stop Solution (Part No. 60193): Two 12 mL bottles containing 0.5M HCl.
      View All» UniProt Number:
      • P19838

      • Q00653

      • Q04206

      • Q04864

      • Q01201
        View All» Packaging: 2 x 96 wells per plate View All» Entrez Gene Number: NM_003998.2 View All»
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