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.
