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  • 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