Product Datasheet. Secreted Alkaline Phosphatase Reporter Kit NBP Unit Size: 1 Kit. Storage is content dependent.

Transcription

1 Product Datasheet Secreted Alkaline Phosphatase Reporter Kit NBP Unit Size: 1 Kit is content dependent. Publications: 6 Protocols, Publications, Related Products, Reviews, Research Tools and Images at: /NBP Updated 6/15/2014 v.20.1

2 NBP Secreted Alkaline Phosphatase Reporter Assay Kit Product Information Unit Size Concentration 1 Kit Concentration is not relevant for this product. Please see the protocols for proper use of this product. is content dependent. Page 1 of 6 v.20.1 Updated 6/15/2014 Product Description Immunogen Kit Components This Kit provides sufficient reagents for 400 quantitative measurements of SEAP protein in 96-well microtiter format. All 96 wells can be used at one time or you may use only the wells as needed by your experimental design. 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. Secreted Placental Alkaline Phosphatase standard SEAP (200 ug/ml), PNPP Substrate, Dilution Buffer (10X), PNPP Buffer (10X), 96 well Assay Plates, Plate Sealer Product Application Details Applications Recommended Dilutions Application Notes Images ELISA ELISA ELISA: Secreted Alkaline Phosphatase Reporter Assay Kit [NBP ] - 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. Sampling Ease: 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 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.

3 Publications Page 2 of 6 v.20.1 Updated 6/15/2014 Greene CM, Carroll TP, Smith SG et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol Feb 1 [PMID: ] 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 P0tzsch S, Blankenhorn A, Navarrete Santos A et al. The effect of an AGE-rich dietary extract on the activation of NFkB depends on the cell model used. Food Funct Jul [PMID: ] 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 Jun 15 [PMID: ] 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 Oct 22 [PMID: ] 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 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 Jun 13 [PMID: ] 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/NFkB/SEAPorter HEK 293 cell line (IML-105). 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 Feb 15 [PMID: ] 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

4 Procedures MSDS (NBP ) Product and Contents Product Name Nf-kB SEAPorter Assay Kit, SEAPorter Assay Kit Catalog No. NBP Page 3 of 6 v.20.1 Updated 6/15/2014 Contents p-nitrophenyl Phosphate (KC-103), Secreated Placental Alkaline Phosphatase (IMK-515-1), SEAP Plasmids (IMK-515-3, IMK-515-4), Sodium Azide (Sample Dilution Buffer, IMK-515-6) CAS#: (pnpp, KC-103) CAS#: None (SEAP, IMK-515-1) CAS#: None (SEAP Plasmids IMK-515-3, IMK515-4) CAS# (10X Sample Dilution Buffer with 0.05% Sodium Azide, IMK-515-6) p-nitrophenyl Phosphate (pnpp) KC-103 Skin: Causes skin irritation. May be harmful if absorbed through skin. Ingestion: May be harmful if swallowed. Call a physician Use with adequate ventilation. Minimize dust generation and accumulation. Avoid contact with eyes, skin, and clothing. Avoid ingestion and inhalation. Store at -20 degrees C. Store in a tightly closed container. Light and moisture sensitive. Stable under recommended Skin: Flush skin with excess amounts of water for at least 15 minutes. Consult a physician Ingestion: Toxic material. If swallowed, get medical aid immediately. Only induce vomiting if directed to do so by Secreted Alkaline Phosphatase Skin: Causes skin irritation. May be harmful if absorbed through skin. Ingestion: May be harmful if swallowed. Call a physician Handle as if capable of transmitting infectious agents. Use with adequate ventilation. Avoid contact with eyes, skin, and clothing. Avoid ingestion and inhalation.

5 Store at -20 degrees C. Store in a tightly closed container. Light and moisture sensitive. Stable under recommended Skin: Flush skin with excess amounts of water for at least 15 minutes. Consult a physician Ingestion: POISON material. If swallowed, get medical aid immediately. Only induce vomiting if directed to do so by SEAP Plasmids Skin: Causes skin irritation. May be harmful if absorbed through skin. Ingestion: May be harmful if swallowed. Call a physician Handle as if capable of transmitting infectious agents. Use with adequate ventilation. Avoid contact with eyes, skin, and clothing. Avoid ingestion and inhalation. Store at -20 degrees C. Store in a tightly closed container. Light and moisture sensitive. Stable under recommended Skin: Get medical aid immediately. Immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. SPEEDY ACTION IS CRITICAL! Ingestion: POISON material. If swallowed, get medical aid immediately. Only induce vomiting if directed to do so by 10X Sample Dilution Buffer with 0.05% Sodium Azide Page 4 of 6 v.20.1 Updated 6/15/2014

6 Skin: Causes skin irritation. May be harmful if absorbed through skin. Ingestion: May be fatal if swallowed. Page 5 of 6 v.20.1 Updated 6/15/2014 Use with adequate ventilation. Minimize dust generation and accumulation. Avoid contact with eyes, skin, and clothing. Avoid ingestion and inhalation. Store at 4 degrees C. Store in a tightly closed container. Light and moisture sensitive. Stable under recommended Skin: Get medical aid immediately. Immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. SPEEDY ACTION IS CRITICAL! Ingestion: POISON material. If swallowed, get medical aid immediately. Only induce vomiting if directed to do so by

7 Novus Biologicals USA 8100 Southpark Way, A-8 Littleton, CO USA Phone: Toll Free: Fax: Novus Biologicals Canada 461 North Service Road West, Unit B37 Oakville, ON L6M 2V5 Canada Phone: Toll Free: Fax: Novus Biologicals Europe 19 Barton Lane Abingdon Science Park Abingdon, OX14 3NB, United Kingdom Phone: (44) (0) Free Phone: Fax: (44) (0) General Contact Information Technical Support: Orders: General: 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. For more information on our guarantee, please visit /guarantee.