July 11, 2017

Western Blot Buffers: TBS and PBS

By Cytiva

TBS and PBS are commonly used buffers for various Western blot protocols. Here we consider which to use, and when.

What is a Western blot buffer?

In the Western blot protocols after the transfer from gel to membrane, we need several different solutions for blocking, diluting antibodies and washing. To prepare those solutions, we often use a Western blot buffer, either Phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) to provide “buffering” function to each solution.

Why do we use a blocking buffer?

We do this to detect specific binding of the antibody to the protein of interest, and with high background from nonspecific binding of antibody to the membrane, it masks the signal from the protein of interest, reducing specificity and sensitivity. The blocking step minimizes this background signal by "blocking" spaces on the membrane not already occupied by proteins.

Should I use PBS or TBS for my Western blotting?

PBS and TBS are commonly used buffers for various stages in Western blotting protocols, and it’s useful to know when to use each of them. Most laboratories use buffer systems dictated by long-established standard operating procedures (SOPs). However, the buffer you choose for Western blotting will also depend on your proteins and the antibodies used. So, it’s worth breaking protocol to explore whether PBS or TBS buffers give you the best final signal and least background. If you are experiencing high background issues, click our Western Blotting Troubleshooting Tips.

Many researchers use PBS and TBS as dilution buffers for blocking agents and they often find that PBS and TBS are interchangeable. However, not all membrane blocking buffer formulations are suitable for every situation or antibody and there are instances where PBS cannot be used. For example, PBS interferes with alkaline phosphatase (AP)-conjugated secondary antibody and in this case, you should use TBS instead.

TBS blocking buffers are also the best choice for detecting phosphorylated protein molecules with phospo-specific antibodies. In this case, the primary antibody will not only bind to phosphate on the target protein, but also to that in the PBS buffer, significantly reducing your target signal.

Western blot: membrane washing buffers

After the primary antibody step, wash the membrane to remove excess antibody. This excess can cause high background signal and, consequently, low signal-to-noise ratio.

A low-concentration detergent solution, such as 0.05% to 0.1% Tween™ 20 in PBS or TBS buffer is commonly used for this washing step, especially after incubation with highly concentrated antibody solutions or crude extracts.

Why is Tween 20 used?

PBS-Tween is normally sufficient for most Western blot washing applications, but it’s important to use TBS-Tween where the target proteins are phosphorylated, for the reasons mentioned above. This is a detergent (surfactant) that is often added to buffers for immunohistochemistry and will help you with problems occurring in your application.

Overview: Western blot buffer TBS/PBS

For alkaline phosphatase-based detection and Western blotting of phosphorylated proteins, use TBS-based buffers, but for most other Western blot applications, both PBS and TBS buffers are largely interchangeable. However, don’t be afraid to challenge your SOP -- you might find that using a different buffer works wonders.

For information on how to prepare Western blot buffers, check out our Western Blotting Principles and Methods Handbook.