Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric)

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 added to wells of a 96-microtiter plate and the standard curve was generated according to the Kit assay protocol.

• Applications: Enzyme Activity

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.
• Specificity: 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.
• Kit Type: Assay Kit (Colorimetric)

Applications/Dilutions

• Dilutions:
  • Enzyme Activity

Packaging, Storage & Formulations

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

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.

Publications for Secreted Alkaline Phosphatase Reporter Kit (NBP2-25285)(11)

Publications using NBP2-25285

• Pistikou A, Cremers G, Nathalia B et al. Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells bioRxiv 2023-01-19 [PMID: 37919273] (EnzAct)
• Denolly S, Guo H, Martens M et al. Dengue virus NS1 secretion is regulated via importin-subunit ?1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin mBio 2023-09-13 [PMID: 37702492]
• Mark RB and Marc GA Utility of Alternative Promoters for Foreign Gene Expression Using the Baculovirus Expression Vector System Viruses 2022-11-29 [PMID: 36560674]
  
  Details:
  The supernatants of infections with SEAP-producing rBEVs were harvested by centrifugation at 1000× g for 10 min.
• Lapierre J, Rodriguez M, Ojha CR, El-Hage N Critical Role of Beclin1 in HIV Tat and Morphine-Induced Inflammation and Calcium Release in Glial Cells from Autophagy Deficient Mouse. J Neuroimmune Pharmacol. 2018-05-11 [PMID: 29752681] (Bacteria)
  
  Details:
  Mice were infected with S. pneumoniae.
• Fereig RM, Kuroda Y, Terkawi MA et al. Immunization with Toxoplasma gondii peroxiredoxin 1 induces protective immunity against toxoplasmosis in mice. PLoS ONE. 2017-04-27 [PMID: 28448521] (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. 2013-06-13 [PMID: 23656327]
• 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. 2013-06-15 [PMID: 23558011]
  
  Details:
  Products cited (Fig 5C): 1. RAW NF-kB/SEAP Reporter Cell Line (IML-120) 2. SEAPorter Assay Kit (10055K) Note: The results showed LPS activation of SEAP activity in the IML-120 cells could be attenuated by pre-treating the cells with pre-term milk + neuram
• 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. 2013-07-01 [PMID: 23426622]
  
  Details:
  IMGENEX products cited: 1. RAW NF-kB/SEAP Reporter Cell Line (IML-120): Figs 3, 4 2. SEAPorter Assay Kit (10055K): Fig 3 Note: Bread crumb extract (BCE) was used as an advanced glycation end product to treat RAW IML-120 cells. NF-kB activation was measure
• 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. 2013-02-15 [PMID: 23203159]
  
  Details:
  1. TLR4/NF-kB/SEAPorter HEk 293 cell line (IML-104).
• 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. 2010-10-22 [PMID: 20710027]
• Greene CM, Carroll TP, Smith SG et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol. 2005-02-01 [PMID: 15661927]
  
  Details:
  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 Transfected HEK 293 Cells (IML-107). 5. LPS from E. Coli, TLR4 Ligand (IMG-2204). 6. SEAPorter Assay Kit (10055K).

FAQs for Secreted Alkaline Phosphatase Reporter Kit (NBP2-25285). (Showing 1 - 2 of 2 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.
2. how many 96-well plates can we run with this SEAP assay kit: NBP2-25285
   • This kits comes with 4 plates, 4 plate sealers, and all reagents for 4 x 96-well plates.