PhosphoSerine Western Blotting
Run 40% of IP eluate on 10% gel.
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 rabbit-anti-phosphoserine antibody from millipore in IgG-Free/Protease-Free BSA/TBST (1:2000, you can try 1:1000 if signal is not great at 1:2000).
Probe overnight at 4C in cold room on tilting tray.
Rinse membrane 8x over the course of 2hrs with 1x TBST Block membrane in 5% milk/TBST for 1hr Probe with mouse-anti-rabbit light-chain specific secondary antibody at 1:2000 for 2hrs at RT.
Rinse 6x with TBST over 1hr at RT.
Activate membrane with pico ECL.
Visualize with Amersham film
