Western Blotting: Difference between revisions
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#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 (in TBST) 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 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 (20 ml 2% BSA 1ul of both secondary antibodies) | #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 | ||