Western Blots

Run protein samples in the PCR “Western Denature” 
Load them into gel
Fill appropriately with Running Buffer
 - Fill cassettes all the way
 - Fill Container to the indicated line
Run at 50V  for 1 hour (or until it runs off the stacking gel) and then turn it up to 100V for approx.1 hour
WATCH it until the blue line is at the bottom of the gel and then STOP it (CAREFUL NOT to let it run off the gel)
Cut and soak membrane in methanol for 1 min.
Soak sponges, membranes, blotting paper in transfer buffer until gel is ready
Sandwich gel and membrane between 2 sponges and blotting paper in the cassette. (gel to back)  The order is as follows:
- Back side (black) - sponge - filter paper - gel - membrane - filter paper - sponge - front side
Put cassette in transfer buffer with the ice pack and stir bar.  Fill to the top.
Start run at 30V for 2 hours

Immerse membrane in a small amount of Pons Os (to check if transfer worked)
Rinse under distilled water
Block in TBS milk(5%) for an hour or more
Make Antibody with 5%TBS milk (5ml per membrane if sealed in packet or 10ml if put on the shaker)
Put in sealed packet and spin overnight in fridge or put it in a container on the shaker in the cold room overnight
Rinse 3x in TBS Tween for 5 min each
Put in Secondary Antibody for 1 hr on shaker at room temperature

Troubleshooting your western blot: