Western Analysis (E8023)

Protocol

1. After transfer, incbubate membrane with 25 ml of 1X TBS/T for 5 minutes at room temperature
2. Incubate membrane in 25 ml of Blocking Buffer for 1 hour at room temperature.
3. Incubate membrane in 12 ml of Primary Antibody Solution for 1 hour at room temperature (or overnight at 4°C) with gentle agitation.
4. Wash with 25 ml of TBS/T for 5 minutes. Discard and repeat wash step two more times.
5. Incubate membrane with HRP-conjugated Secondary Antibody Solution for 1 hour at room temperature.
6. Wash with 25 ml of TBS/T for 5 minutes. Discard and repeat wash step two more times.
7. Proceed with detection, using any HRP-compatible chemiluminescent detection kit. 
   
   Solutions Required for Western Analysis:
   
   10X TBS (per liter):  
   24.2g Tris base
   80g NaCl
   Milli-Q™ water
   pH 7.6
   
   1X TBS/T (per liter):
   100 ml 10X TBS
   1 ml Tween-20
   800 ml MilliQ water
   
   Blocking Buffer (in 1X TBS/T):
   2.5% BSA
   2.5% non-fat milk
   
   Primary Antibody Solution:
   1:2000 dilution of Anti-GLuc Antibody in Blocking Buffer
   
   HRP-conjugated Secondary Antibody Solution:
   1:2000 dilution of Anti-rabbit IgG in Blocking Buffer