Western Analysis (E8023)
Protocol
- After transfer, incbubate membrane with 25 ml of 1X TBS/T for 5 minutes at room temperature
- Incubate membrane in 25 ml of Blocking Buffer for 1 hour at room temperature.
- Incubate membrane in 12 ml of Primary Antibody Solution for 1 hour at room temperature (or overnight at 4°C) with gentle agitation.
- Wash with 25 ml of TBS/T for 5 minutes. Discard and repeat wash step two more times.
- Incubate membrane with HRP-conjugated Secondary Antibody Solution for 1 hour at room temperature.
- Wash with 25 ml of TBS/T for 5 minutes. Discard and repeat wash step two more times.
- 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