Hello everybody,

We are trying to thiolate the antibody in order to couple it to the liposomes via a maleimide group (present of the pegylated phospholipid). The thiolation itself is carried out with the Traut's reagent. To minimize the number of possible fails we decided to confirm the presence of free sulfhydryls by reaction with fluoresceinyl-maleimide, for which we have an optimized labeling procedure. Till now we were not succesfull. Can anybody suggest any change/improvement in the thiolation protocol we have? here it is:

a) 0.2 mg antibody + 10-fold molar excess of Traut, phosphate buffer pH 8, degassed with N2, contains 5mM EDTA. Reaction 1h, room temp.

b) purification+buffer exchange using desalting column to phosphate buffer pH 6.5 (degassed)

c) labeling reaction with 2ug of fluoresceinyl-maleimide (stock 1mg/mL in DMSO). reaction 30min, room tepm.

d) purification+buffer exchange using desalting column to PBS pH 7.4. UV-VIS analysis

We are thinking maybe about adding EDTA to the buffer in the steb b) and increasing the amount of Traut. We will be very gratefull for any comments and advices. Thank you.

Denisa