Gal-T Conditions according to Catherine for big plates

For the Gal-T experiment, beads are further washed 2X in 15 mM Tris pH 7.4 (PMSF+Aprotinin). Then, remove as much fluid as possible using a 30-G needle.

 

Quantities of antibody to do an IP from a 100 mm plate.

anti-Flag : 4 µl

anti-Myc : 15 µl

5172 : 54 µl

I have checked that it is no need tu use Ram (Rabbit anti-Mouse antibody) to link the beads to these antibodies

Quantity of beads to do an IP from a 100 mm plate :

30-50 µl (1:1 in PBS + PMSF + Aprotinin)