Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric)

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Enzyme Activity: Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric) [NBP2-25285] - Secreted Alkaline Phosphatase Reporter Assay Kit [NBP2-25285] - Standard curve. A serial dilution of SEAP protein was ...read more

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Summary
Applications Enzyme Activity

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Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric) Summary

Description
This Kit provides sufficient reagents for 400 quantitative measurements of SEAP protein in 96-well microtiter format.
  • Multiple samples can be analyzed in a low-volume, high through-put experiement.
  • Full analysis complete in under 1 hr.
  • SEAP is secreted into the media so no cell lysis is required for the detection of its enzymatic activity.
  • A secreted indicator function permits multiple kinetics experiments using only one culture through sequential sampling of the medium, and also allows the cells to be used for other purposes, such as RNA extraction, Western blot analysis, and other assays.
  • Heating samples at 65C for 10-30 mins can destroy endogenous alkaline phosphatase but not SEAP.
  • SEAP is stable in serum, allowing it to be used as a reporter assay in vivo. SEAP catalyzes the hydrolysis of pNitrophenyl phosphate (PNPP) producing a yellow product that can be read in a spectrophotometer or ELISA reader at 405 nm.
  • Kit Type
    Assay Kit (Colorimetric)

    Applications/Dilutions

    Application Notes
    Use of duplicate wells for the SEAP standard is recommended to obtain accurate results. This kit can be used with supernatant from transfected cells as described or serum as determined by your experimental protocol.
    Publications
    Read Publications using
    NBP2-25285 in the following applications:

    Packaging, Storage & Formulations

    Storage
    Storage of components varies. See protocol for specific instructions.

    Kit Components

    Components
    1. Microplate Seal (4 EA)
    2. 96-well Microplate (4 EA)
    3. 10X Sample Dilution Buffer (10 mL)
    4. 10X PNPP Buffer (4 mL)
    5. SEAP Standard at 200 ug/mL (10 uL)
    6. PNPP Substrate (8 x 5 mg)

    Alternate Names for Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric)

    • SEAP assay

    Background

    Placental alkaline phosphatase (PLAP) is one of the most stable isoenzymes, only existing in the placenta of higher primates. These characteristics make placental alkaline phosphatase suitable to use as a reporter gene for the analysis of promoter activity and gene expression in cell culture and animal serum. The natural form of PLAP is membrane anchored. The recombinant form of placental alkaline phosphatase (secreted alkaline phosphatase, SEAP) is used for reporter gene function. SEAP is designed by inserting a translational terminator after amino acid 489 (Berger, et al., Gene 66 (1): 10 (1988). This mutation converts the membrane-bound PLAP protein into the secreted protein SEAP.

    Limitations

    This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.
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    Publications for Secreted Alkaline Phosphatase Reporter Kit (NBP2-25285)(7)

    We have publications tested in 1 confirmed species: Mouse.

    We have publications tested in 1 application: ELISA.


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    Showing Publications 1 - 7 of 7.
    Publications using NBP2-25285 Applications Species
    Fereig RM, Kuroda Y, Terkawi MA et al. Immunization with Toxoplasma gondii peroxiredoxin 1 induces protective immunity against toxoplasmosis in mice. PLoS ONE. Apr 27 2017 [PMID: 28448521] (ELISA, Mouse) ELISA Mouse
    Chan M, Hayashi T, Mathewson RD et al. Identification of substituted pyrimido[5,4-b]indoles as selective Toll-like receptor 4 ligands. J Med Chem. 2013 Jun 13 [PMID: 23656327]

    Details:
    Products cited (Fig 2A): 1. TLR2/NF-kB/SEAPorter HEK 293 cell line (IML-102) 2. TLR3/NF-kB/SEAPorter HEK 293 cell line (IML-103) 3. TLR4/MD2/CD14/NF-kB/SEAPorter HEK 293 cell line (IML-104MD2CD14) 4. TLR5/NF-kB/SEAPorter HEK 293 cell line (IML-105).
    Namachivayam K, Blanco CL, Frost BL et al. Preterm human milk contains a large pool of latent TGF-B, which can be activated by exogenous neuraminidase. Am J Physiol Gastrointest Liver Physiol. 2013 Jun 15 [PMID: 23558011]
    Potzsch S, Blankenhorn A, Navarrete Santos A et al. The effect of an AGE-rich dietary extract on the activation of NF-kB depends on the cell model used. Food Funct. 2013 Jul [PMID: 23426622]
    Ahmed I, Roy B, Chandrakesan P et al. Evidence of functional cross talk between the Notch and NF-kB pathways in nonneoplastic hyperproliferating colonic epithelium. Am J Physiol Gastrointest Liver Physiol. 2013 Feb 15 [PMID: 23203159]

    Details:
    Products cited (Fig 4B): 1. TLR4/NF-kB/SEAPorter HEK 293 cell line (IML-104) 2. SEAPorter Assay Kit (10055K) Note: Citrobacter rodentium (CR) infected IML-104 TLR4 cells had increased SEAP activity compared to control cells. CR a gram-negative, noninvasiv
    Chandrakesan P, Ahmed I, Anwar T et al. Novel changes in NF-{kappa}B activity during progression and regression phases of hyperplasia: role of MEK, ERK, and p38. J Biol Chem. 2010 Oct 22 [PMID: 20710027]

    Details:
    Products cited (Fig 5A & 5B): TLR4/NF-kB/SEAPorter HEK 293 (IML-104) cell line and the SEAPorter Assay Kit (10055K). Functional Assay: The HEK 293 TLR4 (IML-104) cell line was infected with citrobacter rodentium. SEAP activity was assayed in both the cell
    Greene CM, Carroll TP, Smith SG et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol. 2005 Feb 1 [PMID: 15661927]

    Details:
    Products cited (Functional Assays):1.TLR2/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-102). 2. TLR4/MD-2/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-104MD2). 3. TLR4/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-104).4.TLR7/NF-kB/SEAP Stably

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    FAQs for Secreted Alkaline Phosphatase Reporter Kit (NBP2-25285). (Showing 1 - 1 of 1 FAQs).

    1. Could you tell us if Secreted Alkaline Phosphatase Reporter Kit (Cat#NBP2-25285) contains recombinants? If yes, please let us know expression system of the recombinants.
      • The Secreted Alkaline Phosphatase Reporter Kit contains one protein standard. This standard is purified human native protein.

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