Difference between revisions of "Western Blotting"

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## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
 
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
 
#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.   
 
#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).
+
#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
−
#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)
+
#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished 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)
 
#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
 
#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 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 (2g BSA in 100ml TBST, stored in fridge) for 1 hour
 
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
−
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h  
+
#Incubate with primary antibody (check for dilution) in 2% BSA for >1h  
 
#Wash blot every 5 minutes for 15 min with TBST.
 
#Wash blot every 5 minutes for 15 min with TBST.
 
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
 
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)

Revision as of 15:52, 11 August 2017

Materials

  • Transfer Buffer (200 mL Methanol, 100 mL 10X Transfer Buffer to final 1L volume)
  • Transfer Apparatus, either Bio-Rad or Invitrogen

Protocol

  1. Run SDS-PAGE gel using SDS-PAGE Running Buffer and prepare diluted transfer buffer
    1. Use a prepared 5-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out.
    2. Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
    3. Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
    4. Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
  2. 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.
  3. Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
  4. Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
  5. Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
  6. Scan using licor for total protein, which will be used to normalize the blot
  7. 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)
  8. Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
  9. Incubate with primary antibody (check for dilution) in 2% BSA for >1h
  10. Wash blot every 5 minutes for 15 min with TBST.
  11. Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
  12. Wash blot every 5 minutes for 15 min with TBST.
  13. Rinse once or twice with double distilled water
  14. 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).
  15. Drain excess buffer from blot and cover with ECL for about a minute
  16. Drain excess ECL from blot, cover with saran wrap and expose film

If Using LiCor

  1. Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan
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