PhosphoThreonine Western Blotting
Run 40 % of IP eluate on 10 % gel Tris - Glycine .
Transfer protein to PVDF membrane for 1hr at RT with ice pack ( 100V ) .
Cut membrane just below 180kD ( top MW marker on benchmark prestained ladder ) .
Cut Membrane at ~ 82kD ( the blue band above the pink band using benchmark prestained ladder ) .
* * Note , the GST - tagged protein is ~ 115 kD .
Cutting the membrane in the places will help reduce non - specific binding .
Also , running a ladder in the middle of the gel will help you cut straight across the membrane in the event that the gel is transferred at an angle relative to the membrane .
Block membrane with 5 % BSA ( IgG and Protease - Free ) / TBST for 2hrs at RT .
Probe membrane with phospho - threonine - HRP antibody in IgG - Free / Protease - Free BSA / TBST ( 1 : 1000 ) .
Probe overnight at 4C in cold room on tilting tray .
Rinse membrane 8x over the course of 2hrs with 1x TBST .
Activate membrane with pico ECL .
* * Note , the anti - pThr antibody is HRP conjugated so you don't need to use a secondary .
