Dear Innocent Damudu Peter,

you can use either the Carbodiimid method (with NHS) or generate a mixed carboxylic acid anhydrid with iso butyl chloroformate. I recommend the Carbodiimide/NHS method.

Therefore, dissolve the pregnanolone hemisuccinate either in dry THF or dry DMF at a concentration of 0.17 mol/L (Use e.g. 2.1 mg (5µmol) and dissolve in 30µL) make sure it is completely dissolved. Add 1.2 eq (6µmol) of Sulfo-NHS and DCC from a 0.5 mol/l stock solution in either THF or DMF, whatever you used before. (in the example here: add 15µL Sulfo-NHS, 15µL DCC). Add a spatula tip of about 1 mg of Disuccinimidyl Carbonate DSC. Vortex, until everything is dissolved and shake over night at RT. Centrifuge to remove precipitated Dicyclohexylurea. Use the supernatant for coupling to BSA without further clean up.

Try Adding the NHS-Ester in different molar ratios to BSA: (1:30; 1:50; 1:100). Dissolve the BSA in carbonate buffer (pH = 8.5) Add the NHS-ester slowly dropwise. If you add eg. 30µL add 3 times 10µL dropwise and wait 5 minutes, then add another 10 µL and so on.

Shake this for 3-5 h

Clean up the conjugate via Sephadex column chromatographie (gel chromatographie) Use a Bradford assay to determine the cocnentration of the conjugate, Use MALDI-MS to confirm the coupling and check the coupling ratios.

Use gloves and a hood all the time, the chemicals are toxic and easy flammable!

A Reference is attached!

Article Comparative characterization of mAb producing hapten-specifi...

Good luck!

Peter

P.S.

Fore sure you need to optimize the coupling. The protocols for NHS-ester preparation should do it, I used it for carboxylic acid derivatives of Ethinlyestradiol and Estrone as well, and they have similar structures. but the coupling conditions for BSA maybe need to be optimized. (pH; Coupling time, temperature, amount of NHS-ester and so on...) You also have to decide, whether you go for Sulfo-NHS, which makes the molecule more buffer soluble, then pure NHS, but sometimes the coupling is slower.

Dear Peter Carl,

i just cant thank you enough..

sincerely grateful

Innocent