Protocol for proinsulin biosynthesis by 35S pulse labeling-Protocol generously provided by Dr. Constantin Polychronakos and Dr. Xiaoyu Du, McGill University Health Centre Montreal, Canada.
1. Culture INS-1 cells to 80% confluency.
2. Change to medium without methionine & cystine for 1 hour.
3. Change to fresh medium without methionine & cystine, add 35S methionine & cystine for 30 min.
4. Lyse cells.
1. Add proinsulin antibody NB100-73011 into cell lysate. Use mouse monoclonal antibody as control. Incubate at 4 degrees C overnight.
2. Add protein A agarose, incubate 4 degrees C for 4 hours.
3. Wash beads 3 times in PBS.
4. Elution in 2X loading buffer.
16% /6M separating gel, 10% spacer gel and 4% stacking gel. (Nature protocols, 2006, 1(1),16)
Current protocols in Molecular Biology Appendix 3.