Western Blotting
Materials
- Transfer Buffer (200 mL Methanol, 100 mL 10X Transfer Buffer to final 1L volume)
- Transfer Apparatus, either Bio-Rad or Invitrogen
Protocol
- Run SDS-PAGE gel using SDS-PAGE Running Buffer and prepare diluted transfer buffer
- Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
- Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
- Stain for total protein with Revert total protein stain (let sit for 5 minutes, after 5 minutes pour total protein stain back in bottle for later use)
- Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
- Scan using licor for total protein, which will be used to normalize the blot
- Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
- Rinse nitrocellulose in 2% BSA for 1 hour
- Carefully remove blot, stain with Ponceau solution and rinse with TTBS until all the red is washed off
- Block with 2% BSA in TBST or 5% skim milk powder in TBST for >1h
- Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
- Wash blot every 5 minutes for 15 min with TBST.
- Incubate with appropriate secondary antibody (10 000X) for 45min-1h
- Wash blot every 5 minutes for 15 min with TBST.
- Rinse once or twice with double distilled water
- Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
- Drain excess buffer from blot and cover with ECL for about a minute
- Drain excess ECL from blot, cover with saran wrap and expose film
If Using LiCor
- Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan