Difference between revisions of "Western Blotting"

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==SOP==
 +
*Irritant
 +
*Electrophoresis
 +
 
==Materials==
 
==Materials==
 
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
 
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
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==Protocol==
 
==Protocol==
 +
#Turn on heat block to 85 degrees
 
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
 
#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.   
+
## Use a prepared 4-12% 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 and rinse with water.
−
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
+
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
−
#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)
+
##Boil sample at 85 degrees for ~3 min
−
#Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
+
##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.
 +
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel. (Tip: Gel runs more evenly if you start a lower V and increase once the samples have run down 1/3 of the gel.)
 +
#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), 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 (room temp with an ice pack).
 +
#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!
 +
#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic 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 for 1 hour
+
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
−
#Carefully remove blot, stain with Ponceau solution and rinse with TTBS until all the red is washed off
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#Incubate with primary antibody (check for dilution) in 2% BSA for >1h  
−
#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.
 
#Wash blot every 5 minutes for 15 min with TBST.
−
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h
+
#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)
 
#Wash blot every 5 minutes for 15 min with TBST.
 
#Wash blot every 5 minutes for 15 min with TBST.
−
#Rinse once or twice with double distilled water
+
#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).
+
#Scan dry blot using the LiCor Odyssey [[Scanning and Analyzing Western Blots Using LiCor Odyssey]].
−
#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==
 
==If Using LiCor==
−
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan
 
  
 +
[[Scanning and Analyzing Western Blots Using LiCor Odyssey]]
  
  
 
[[ Category: Western Blotting ]]
 
[[ Category: Western Blotting ]]

Latest revision as of 15:58, 26 March 2021

SOP

  • Irritant
  • Electrophoresis

Materials

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

Protocol

  1. Turn on heat block to 85 degrees
  2. Run SDS-PAGE gel using SDS-PAGE Running Buffer and prepare diluted transfer buffer
    1. Use a prepared 4-12% 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 and rinse with water.
    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. Boil sample at 85 degrees for ~3 min
    4. Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.
    5. Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel. (Tip: Gel runs more evenly if you start a lower V and increase once the samples have run down 1/3 of the gel.)
  3. Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), 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.
  4. Transfer 4h at 75V (in cold room) or overnight at 35V (room temp with an ice pack).
  5. 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!
  6. Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water)
  7. Scan using licor for total protein, which will be used to normalize the blot
  8. 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)
  9. Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
  10. Incubate with primary antibody (check for dilution) in 2% BSA for >1h
  11. Wash blot every 5 minutes for 15 min with TBST.
  12. 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)
  13. Wash blot every 5 minutes for 15 min with TBST.
  14. Rinse once or twice with double distilled water.
  15. Scan dry blot using the LiCor Odyssey Scanning and Analyzing Western Blots Using LiCor Odyssey.

If Using LiCor

Scanning and Analyzing Western Blots Using LiCor Odyssey

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