货号：70-515

品牌：Merck Millipore

规格：

目录价：￥8096.00

市场价格：￥6881.60

会员价格：￥6476.80

金山科研平台，产品价格货期咨询微信：jinshanbio Description: NFκB p50 Transcription Factor Assay View All» Trade Name: Upstate (Millipore) View All» Qty/Pk: 1 plate View All» Product Overview: Test Principle The Non-Radioactive NFkB p50 Transcription Factor Assay kit is provided in a 96-well format. During the assay, the Capture Probe, a double stranded biotinylated oligonucleotide containing the consensus sequence for NFkB binding (5'-GGGACTTTCC-3'), is mixed with cellular (nuclear) extract in the Transcription Factor Assay Buffer provided. When incubated together, the active form of NFkB contained in the nuclear extract binds to its consensus sequence. After incubation, the extract/probe/buffer mixture is then directly transferred to the streptavidin coated plate. The biotinylated double stranded oligonucleotide bound by active NFkB protein is immobilized and any inactive, unbound material is washed away. The bound NFkB transcription factor subunit, p50, is detected with a specific primary antibody, a Rabbit anti-NFkB p50 (active form). An HRP-conjugated secondary antibody is then used for detection and provides sensitive colorimetric detection that can be read in a spectrophotometric plate reader. Included in the kit are positive cell extract, a non-specific double stranded oligonucleotide, and a specific competitor double stranded oligonucleotide. The NF kappa B Transcription Factor Assays were QC tested using nuclear extracts from human (HeLa) cells. However, because of the conservation in the NFkB DNA binding site and the fact that the antibodies contained in the kit cross react with rat and mouse NFkB, the kit is expected to work with samples from rat and mouse as well as human. For Research Use Only; Not for use in diagnostic procedures Use of this assay in NFkB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA). View All» Key Applications: Activity Assay View All» Application Notes: The transcription factor NFkB (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 repeats. NFkB 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, NFkB1 (p50 and its precursor p105), and NFkB2 (p52 and its precursor p100). NF-kB/Rel proteins exist as homo- or heterodimers to form transcriptionally competent or repressive complexes. Although most NFkB 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 NFkB is the most abundant in cells. A critical component in NFkB regulation is the IkB Kinase (IKK) complex. In a majority of unstimulated cells, the NFkB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IkB proteins. Upon stimulation by multiple inducers including viruses or cytokines, such as TNFa, IL-1, or PMA, IkBa is rapidly phosphorylated and degraded, resulting in the release of the NFkB 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. NFkB induces the transcription of its own inhibitor, IkBa, causing an autoregulatory mechanism of NFkB activity and generating the inactive form of NFkB. The newly formed nuclear NF-kB-IkBa complexes are then exported out to the cytoplasm, thereby reestablishing the cytoplasmic pool of inactive NFkB complexes primed for another round of activation to take place. The wide variety of genes regulated by NFkB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes. The Upstate® Non-Radioactive NFkB p50 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 significantly 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. 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: This gene encodes a 105 kD protein which can undergo cotranslational processing by the 26S proteasome to produce a 50 kD protein. The 105 kD protein is a Rel protein-specific transcription inhibitor and the 50 kD protein is a DNA binding subunit of the NF-kappa-B (NFKB) protein complex. NFKB is a transcription regulator that is activated by various intra- and extra-cellular stimuli such as cytokines, oxidant-free radicals, ultraviolet irradiation, and bacterial or viral products. Activated NFKB translocates into the nucleus and stimulates the expression of genes involved in a wide variety of biological functions. Inappropriate activation of NFKB has been associated with a number of inflammatory diseases while persistent inhibition of NFKB leads to inappropriate immune cell development or delayed cell growth. View All» UniProt Summary: FUNCTION: SwissProt: P19838 # Appears to have dual functions such as cytoplasmic retention of attached NF-kappa-B proteins and generation of p50 by a cotranslational processing. The proteasome-mediated process ensures the production of both p50 and p105 and preserves their independent function, although processing of NFKB1/p105 also appears to occur post-translationally. p50 binds to the kappa-B consensus sequence 5'-GGRNNYYCC-3', located in the enhancer region of genes involved in immune response and acute phase reactions. Plays a role in the regulation of apoptosis.SIZE: 968 amino acids; 105356 Da SUBUNIT: Active NF-kappa-B is a heterodimer of an about 50 kDa DNA-binding subunit and the weak DNA-binding subunit p65. Two heterodimers might form a labile tetramer. Also interacts with MAP3K8. NF-kappa-B p50 subunit interacts with NCOA3 coactivator, which may coactivate NF-kappa-B dependent expression via its histone acetyltransferase activity. Interacts with DSIPI; this interaction prevents nuclear translocation and DNA-binding. Interacts with SPAG9 and UNC5CL.SUBCELLULAR LOCATION: Nucleus. Cytoplasm. Note=Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B).DOMAIN: SwissProt: P19838 The C-terminus of p105 might be involved in cytoplasmic retention, inhibition of DNA-binding, and transcription activation. & Glycine-rich region (GRR) appears to be a critical element in the generation of p50.PTM: While translation occurs, the particular unfolded structure after the GRR repeat promotes the generation of p50 making it an acceptable substrate for the proteasome. This process is known as cotranslational processing. The processed form is active and the unprocessed form acts as an inhibitor (I kappa B-like), being able to form cytosolic complexes with NF-kappa B, trapping it in the cytoplasm. Complete folding of the region downstream of the GRR repeat precludes processing. & Polyubiquitination seems to allow p105 processing.SIMILARITY: Contains 7 ANK repeats. & Contains 1 death domain. & 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 HeLa Whole Cell Extract at -80°C. Avoid repeated freeze-thaw cycles. Stable at -80°C for 6 months. · Store the NFkB Capture Probe, Competitor Oligonucleotides, and Negative Control Probe at -20°C. May be stored at 4°C for less than one month. · Store the Transcription Factor Assay (TFA) Buffer at -20°C. Avoid freeze-thawing of the TFA Buffer. Buffer can be aliquotted. · Store all other components at 4°C. View All» Replaces: SGT515 View All» Gene Symbol:
  • NFKB1

  • NF-kappa-B

  • NFKB-p105

  • EBP-1

  • KBF1

  • DKFZp686C01211

  • MGC54151

  • NFKB-p50
    View All» Product Name: NFκB p50 Transcription Factor Assay View All» Materials Required but Not Delivered: 1. Nuclear Extraction kit or components 2. Pipettor (both single and multi-channel) 3. Pipette tips 4. Distilled water 5. Microplate reader (wavelength 450nm/650nm) 6. Rotator or Plate shaker (optional) 7. Plate cover (optional) View All» Components:
    • Transcription Factor Assay Plate (Part No. 90268): One 96-well Streptavidin coated plate sealed in a foil pouch with desiccant.

    • TFA Buffer, 5x (Part No. 90272): One bottle containing 80 mL of 5x Transcription Factor Assay Buffer containing sonicated salmon sperm DNA to block non-specific DNA binding activity.

    • Blocking Reagent (Part No. 90273): One vial containing 12 grams of Blocking Reagent

    • Blocking Reagent (Part No. 90273): One vial containing 12 grams of Blocking Reagent

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

    • Gt X Rb, HRP Secondary Antibody (Part No. 90276): One vial containing 15 µL Goat anti-Rabbit, HRP conjugated secondary detection antibody. Use at 1:1000 dilution in Enhanced Transcription Factor Assay Buffer.

    • NFkB Capture Probe (Part No. 90269): One vial containing 110 µL biotinylated double stranded oligonucleotide probe (2 pmol/µL) containing the wild-type consensus sequence (5'-GGGACTTTCC-3') for NFkB.

    • NFkB Specific Competitor Oligonucleotide (Part No. 90270): One vial containing 20 µL (10 pmol/µL) specific competitor. The NFkB Competitor Oligonucleotide has the same consensus sequence (5'-GGGACTTTCC-3') as the NFkB 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. 90271): One vial containing 20 µL biotinylated double stranded oligonucleotide probe (2 pmol/µL). Provided as an internal negative control, this non-specific competitor oligonucleotide does not contain the NFkB consensus sequence.

    • HeLa Whole Cell Extract, TNFalpha Treated (Part No. 90350): One vial containing 25 µL TNFalpha 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.

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

    • Stop Solution (Part No. 60193): One 12mL bottle containing 0.5M HCl.

    • (Caution: Caustic material. Eye, hand, face, and clothing protection should be worn when handling this material.)
      View All» UniProt Number: P19838 View All» Packaging: 96 wells View All» Entrez Gene Number: NM_003998.2 View All»
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