Western Blotting: Difference between revisions

Line 10:

## 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.

#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)

#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 (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.

#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)

@@ Line 10: / Line 10: @@
 ## 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.
-#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)
 #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 (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.
 #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)