Mucin biotinylation - NHS - biotin reagent
Calculate how much NHS - Biotin that is needed for biotinylation of the desired amount of mucin ( assumed to have Mw 500 000 Da ) and the molar ratio of biotin : mucin should be 1 : 2000 .
Prepare Phosphate Buffered Saline ( PBS ) with pH 7.4 .
Dissolve mucin in PBS to a concentration of 2mg / ml .
Take out the NHS - Biotin reagent and equilibrate it in room temperature before opening the vial .
Dissolve NHS - Biotin in dimethylsulfoxide ( DMSO ) to a concentration of 10 mM .
Add the pre - calculated volume of biotin reagent to the protein solution and incubate at 4 °C for 4 hours .
To remove unbound biotin , exchange the buffer for water by overnight dialysis .
Transfer the solution to another tube , freeze and freeze - dry .
