反应详情

A 1.25 ml aliquot of a stock solution (1.009 g of (6aR,10aR)-4-[(6a,7,10,10a-Tetrahydro-6,6,9-trimethyl-3-pentyl-6H-dibenzo[b,d]pyran-1-yl)oxy]butanoic acid and 1.00 ml of 25% aqueous tetramethyl ammonium hydroxide diluted to 10 ml with water) was mixed with 8.75 ml of N,N-dimethyl formamide (DMF, Fisher, Lot 761531) to give a final volume of 10 ml with a concentration of 12.6 mg/ml. Two hundred fifty mg of Bovine Serum Albumin (BSA-Pentex-Miles Labs. Lot 260) was dissolved in 25.0 ml of distilled water and 2.5 ml of DMF. Five ml of the above solution was slowly added while stirring to the BSA solution. This solution became slightly turbid, and 6.3 ml of distilled water was added to decrease the turbidity. The remaining 5.0 ml of the above solution was slowly added. Turbidity developed again, and 6.0 ml of distilled water was added. The resultant total volume of the above solution was 49.8 ml. To this 49.8 ml of solution, 0.2 ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDCI; 500 mg/ml solution) was added, giving a final volume of 50.0 ml. The pH of the final solution was 8.4. A beaker with the 50.0 ml of final solution was wrapped in aluminum foil and placed on a magnetic stirrer in a cold room (5° C.) for 24 hrs. The solution was transferred to a dialysis bag (Spectrum Medical Industries Spectrapor #1) and dialyzed against 25% DMF-75% H2O (5x) and against distilled water (2x) for four days in the cold room. Total volume recovered after dialysis was 51 ml. The dialyzate was tested by immunoelectrophoresis using IEP buffer (0.05 ionic strength, pH 8.6 buffer-5.4 g sodium diethyl barbiturate, 2.6 g sodium acetate.3H2O, 58.2 ml 0.1 N hydrochloric acid, 942 ml distilled water); 1% agarose (Kallestad) in IEP buffer; 0.1% bromophenol blue in IEP buffer and goat anti-BSA serum and a precipitin band indicated that the immunogen was different from BSA.

1. customto give a final volume of 10 ml with a concentration of 12.6 mg/ml
2. additionwas added
3. customgiving a final volume of 50.0 ml
4. customdialyzed against 25% DMF-75% H2O (5x)
5. distillationagainst distilled water (2x) for four days in the cold room
6. customTotal volume recovered
7. customafter dialysis
8. distillationbuffer (0.05 ionic strength, pH 8.6 buffer-5.4 g sodium diethyl barbiturate, 2.6 g sodium acetate.3H2O, 58.2 ml 0.1 N hydrochloric acid, 942 ml distilled water)