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ELISA Assays
Vascular Endothelial Growth Factor (VEGF)
Also known as:
- Vascular permeability factor |
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ELISA was done using a kit from R&D Systems (Quantikine mouse VEGF #MMV00). The assay is a direct sandwich ELISA assay. Rat recombinant VEGF (BioSource PRG0115), used in a standard dilution series (curve shown), corroborates with the ELISA response to a dilution series of BN Rat VEC media and extraction. Assay viability is confirmed. The experiment was performed according to the manufacturer’s instructions. |
Vascular Endothelial Growth Factor Receptor1 (VEGF-R1)
Also known as:
- Tyrosine-protein kinase receptor FLT
- FLT-1 |
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The ELISA for Flt 1 was done using an indirect ELISA that was developed in our laboratory. The ELISA response to a dilution series of Brown Norway (BN) Rat VEC extraction is shown. The dilutions series samples are described by their total protein amount, determined by BCA assay (Pierce #23235). Anti-Flt1 was obtained from Santa Cruz Biotechnology Inc. (Flt-1 (c-17): sc-316 rabbit polyclonal IgG). The conjugate antibody was obtained from Pierce (Anti-Rabbit IgG-HRP #31460). |
Vascular Endothelial Growth Factor Receptor2 (VEGF-R2)
Also known as:
- VEGFR-2
- Protein-tyrosine kinase receptor flk-1
- Fetal liver kinase 1 |
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The ELISA assay for Flk-1 was done using an indirect ELISA that was developed in house. The ELISA response to a dilution series of BN Rat VEC extraction is shown. The dilutions series samples are described by their total protein, determined by BCA assay. The anti-Flk-1 antibody used is a mouse monoclonal anti-human Flk-1 antibody obtained from Alpha Diagnostic International. The conjugate antibody was obtained from Pierce. (Anti-Rabbit IgG-HRP #31460).
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Osteopontin (OSTP)
Also known as:
- Bone sialoprotein I
- Secreted phosphoprotein I
- SPP |
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The ELISA was done using a commercially available kit (TiterZyme EIA rat Osteopontin #900-089) obtained from Assay Designs Inc. The same response relation is seen with a dilution series of BN Rat VEC media and extraction, confirming viability. |
Endothelial Nitric-Oxide Synthase (eNOS)
Also known as:
- EC-NOS
- NOS, type III
- NOSIII
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Shown is an ELISA standard curve reproduced from the R&D kit (Quantikine human eNOS #DEN00). In our initial experiments with rVEC lysates, we do not see a similar response, possibly due to low concentrations of the protein in the extract. Alternatively, we have been able to show that two antibodies, a mouse monoclonal Anti-eNOS (BIOMOL # SA-258) and a rabbit polyclonal Anti-eNOS (BD Transduction Laboratories # 610299) cross-react with rat eNOS; and we intend to test rVEC extracts with these antibodies to verify the initial ELISA. Results. |
| Immunoprecipitation |
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For our initial experiments to establish a protocol for immunoprecipitation, we used the Protein Tyrosine Phosphatase 1 beta (PTP-1B) as target protein. Immunoprecipitation of PTP-1B was conducted using human lymphocyte cells (GM15226) obtained from Coriell Cell Repositories (Camden, NJ). Cells were cultured in RPMI-1640 and frozen in aliquots of approximately 1x107 cells. Cells were thawed and lysed using NP-40 lysis buffer. Protein concentration was measured using the Micro BCA Protein Assay Kit (Pierce Biotechnology). Immunoprecipitation was done using the Seize X Mammalian Immunoprecipitation Kit (Pierce Biotechnology) with a monoclonal Mouse IgGa against PTP-1B obtained from BD Transduction Laboratories. Presence of PTP-1B was detected using SDS-PAGE and Western Blotting using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). The block arrow on the right indicates the approximate size of PTP-1B. |
Western Blot analysis of PTP-1B before and after Immunoprecipitation.
Lane 1: Lymphoblast cell lysate
Lane 2-5: series of washes after protein G coupled antibody gel was incubated with cell lysate
Lane 6-8: Immunoprecipitation fractions. |
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