Difference between revisions of "Western Blotting"
From Bridges Lab Protocols
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==Protocol== | ==Protocol== | ||
#Run SDS-PAGE gel and prepare transfer buffer | #Run SDS-PAGE gel and prepare 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 or overnight at 35V. | + | #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). | ||
#Carefully remove blot, stain with Ponceau solution and rinse with TTBS until all the red is washed off | #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 | #Block with 2% BSA in TBST or 5% skim milk powder in TBST for >1h | ||
Revision as of 00:54, 2 May 2009
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 and prepare 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).
- 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 five minutes for 30 min with TBST.
- Incubate with appropriate secondary antibody (10 000X) for 45min-1h
- Wash blot every five minutes for 30 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
