Mouse VEGF ELISA Assay
THe Mouse VEGF ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Vascular Endothelial Growth Factor, VEGF-A, Vascular Permeability Factor, VPF
SAMPLE COLLECTION AND STORAGE
1. Cell Culture Supernates – Remove particulates by centrifugation.
2. Serum – Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum, avoid hemolysis and high blood lipid samples.
3. Plasma – Recommended EDTA as an anticoagulant in plasma. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
4. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.
5. Dilute samples at the appropriate multiple (recommended to do pre-test to determine the dilution factor).
Note: Normal mouse serum or plasma samples are suggested to make a 1:2 dilution.
The Mouse Vascular Endothelial Growth Factor (VEGF) ELISA Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for VEGF has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any VEGF present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for VEGF is added to the wells and binds to the combination of capture antibody-VEGF in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of VEGF present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven VEGF standard dilutions and VEGF sample concentration determined.
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- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µl of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µl) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µl of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
- Repeat the aspiration/wash.
- Add 100 µl of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
- Add 100 µl of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable)
Vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor (VPF), is a potent mediator of both angiogenesis and vasculogenesis in the fetus and adults (1-3). It is a member of the platelet derived growth factor family that is characterized by a cysteine knot structure formed by eight conserved cysteine residues (4). Mouse embryos expressing only the VEGF120 isoform do not survive to term and show defects in skeletogenesis (5). Mouse VEGF120 shares 98% aa sequence identity with corresponding regions of rat, 89% with canine, feline, equine and porcine, and 87% with human, ovine and bovine VEGF, respectively. VEGF binds the type I transmembrane receptor tyrosine kinases VEGF R1 (also called Flt 1) and VEGF R2 (Flk1/KDR) on endothelial cells (4). VEGF is required during embryogenesis to regulate the proliferation, migration, and survival of endothelial cells (3, 4). In adults, VEGF functions mainly in wound healing and the female reproductive cycle (3). Pathologically, it is involved in tumor angiogenesis and vascular leakage (6, 7). Circulating VEGF levels correlate with disease activity in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematous (8).VEGF is induced by hypoxia and cytokines such as IL-1, IL-6, IL-8, oncostatin M and TNF-α (3, 4, 9).
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
1. Ferrara, N. et al. (2003) Nat. Med. 9:669.
2. Shima, D.T. et al. (1996) J. Biol. Chem. 271:3877.
3. Breier, G. et al. (1992) Development 114:521.
4. Claffey, K.P. et al. (1992) J. Biol. Chem. 267:16317.
5. Conn, G. et al. (1990) Proc. Natl. Acad. Sci. USA 87:2628.
6. Scheidegger, P. et al. (1999) Biol. Chem. 380:1449.
7. Sharma, H.S. et al. (1995) Biochim. Biophys. Acta 1260:235.
8. Berse, B. (2002) GenBank Accession # P26617.
9. Keck, P.J. et al. (1989) Science 246:1309.