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Corticosterone-21-thyroglobulin used as the immunogen.
Corticosterone Levels 1. Men 420 ± 220 pg/0.1 ml 2. Women Follicular Phase 240 ± 150 pg/0.1 ml Luteal Phase 440 ± 230 pg/0.1 ml 3. Laying Hens 220 ± 50 pg/0.1 ml.
The antiserum is characterized utilizing the following dextran coated charcoal radioimmunoassay (RIA) protocol, where 0.5 ml of reconstituted and diluted antiserum has been found to bind at least 40% of 15 picograms of tritiated (3H) corticosterone with a specific activity of approximately 100 ci/mmole. RIA Reagents (A) Standards: Prepare a stock standard solution of 1µg/ml corticosterone in absolute ethanol. Dilute a portion of the stock solution with buffer (B) to a concentration of 2,000 pg/0.1 ml. This is further diluted in buffer (B) to obtain standard solutions at the following concentrations: 1000, 500, 250, 125 and 63 pg/0.1 ml. (B) Dilution buffer: 0.05 M Tris-HCl, pH 8.0, containing 0.1 M NaCl, 0.1% BSA and 0.1% sodium azide. (C) Dextran coated charcoal suspension: 0.5% activated charcoal untreated powder 100-400 mesh, 0.5% dextran approximate average molecular weight 70,000 in buffer (B). It is important that the dextran be in solution before the addition of charcoal. The dextran coated charcoal suspension should be stirred and kept at 0 °C in ice-water for at least 30 minutes before and during use. RIA Protocol 1. In polypropylene test tubes, add 0.1 ml sample or standard (A) and 0.5 ml diluted antiserum. 2. Vortex the tubes. 3. Incubate for 30 minutes at room temperature. 4. Add 0.1 ml tritiated radioactive tracer diluted in dilution buffer (B). 5. Vortex the tubes. 6. Incubate for 1 hour at 37 °C. 7. Cool the tubes for 15 minutes at 4 °C. 8. Rapidly add 0.2 ml cold dextran coated charcoal suspension (C) to each tube. 9. Vortex the tubes. 10. Incubate for 10 minutes at 0 °C in ice-water. 11. Centrifuge at 2000 x g for 15 minutes at 4 °C. 12. Remove supernatant from each tube, add scintillation cocktail to the supernatant and determine the amount of radioactivity present. Sensitivity is defined as the 90% intercept of a B/B0 standard curve. In the above system the sensitivity has been found to be 30 picograms/tube. The affinity constant (Ka) is determined by a Scatchard plot using the described RIA system. Ka = 1-10 x 109 L/mole.
Our Abpromise guarantee covers the use of ab10996 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|RIA||Use a concentration of 0.5 mg/ml.|
ab10996 has not yet been referenced specifically in any publications.