How to Block in Western Blot

Western blotting is a technique frequently used in research laboratories.

Western blots can detect the presence of specific proteins in a given sample of tissue extract by using gel electrophoresis to separate the proteins by polypeptide length or 3D structure. After the various proteins have been separated by gel electrophoresis, the proteins are then transferred to a membrane. Antibodies, which bind only to specific proteins, are used to detect the target protein.



Within Western blot, the blocking step reduces the ratio of noise to signal by preventing the occurrence of non-specific binding.

Things You'll Need

• Nitrocellulose or polyvinylidene difluoride membrane containing transferred proteins
• Bovine serum albumin (BSA) or nonfat dry milk
• Tris-buffered saline (TBS) or phosphate buffered saline (PBS)
• Tween-20 or Triton X-100
• Plastic container and cover
• Orbital shaker

Instructions

Preparing the Blocking Solution

1. Prior to blocking the membrane, prepare enough blocking solution to fully cover the membrane when it is placed in the plastic container using one of the following combinations of TBS or PBS, BSA or blotto, and Tween or Triton X-100:

   3 to 5 percent nonfat dry milk and 0.05 to 0.1 percent Tween-20 in PBS or TBS

   1 to 5 percent BSA and 0.05 to 0.1 percent Tween-20 in PBS or TBS

   Use TBS as the buffer solution for phosphorylated proteins.

2. Place the membrane with transferred proteins into the plastic container and carefully pour the prepared blocking solution into the container. Cover the container securely

3. Place the container on a orbital shaker set at a low speed.

4. The container may be left at room temperature for 30 minutes to 2 hours, or it may be refrigerated at 4 degrees Celsius and left overnight.

   After blocking, the membrane will be ready for protein detection via the primary antibody.

Tips & Warnings

• Because blocking for more than 2 hours at room temperature can cause the membrane proteins to be exchanged and lost, make sure to either time or make note of the length of blocking time at room temperature.

• If the final product of the Western blot reveals a great amount of background noise, changing the buffer solution (from TBS to PBS or vice versa) in a second attempt may help reduce that noise.

• As membranes used in Western blots tend to be carcinogenic, use gloves to avoid direct contact when handling membranes.

References

• Millipore: Western Blotting Protocol
• Proteomics: Blocking Non-specific Sites
• Molecular Station: Western Blot
• Proteimax: Western Blot Protocol