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:
http://www.bio-rad.com/en-us/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor