A blot uses five or six buffers and they are easy to confuse, partly because several share the same Tris base and partly because the protocols that name them assume you already know which is which. The transfer buffer is the one with the most argument attached to it, because the methanol in it does two opposite things at once. This page sets out what each buffer is for, and the small number of decisions that actually change a result.
What is in the transfer buffer, and what the methanol does
The classical recipe is Tris, glycine and methanol, and the methanol is the part worth understanding. It strips the detergent off the protein, which is what lets a nitrocellulose membrane bind it, and it shrinks the gel, which keeps the transfer tight. It also slows large proteins down considerably, which is why a protocol for something high in mass often reduces or removes it and adds a little detergent instead. Those are opposite adjustments for opposite problems, so match the buffer to the mass range you care about rather than using one recipe for every blot.
Choosing a western blot stripping buffer, and how often a membrane survives it
Stripping removes bound antibody so the same membrane can be probed again, and the options run from a mild low-pH glycine wash to a reducing detergent buffer that is close to stripping the protein as well. The mild version costs signal on the second probe and keeps the sample; the harsh version reliably clears the antibody and sometimes clears the target with it. Probe for the low-abundance target first, keep an image of every round, and treat more than two or three cycles as a new blot rather than a saved one.
The SDS PAGE running buffer that goes in the tank
The tank buffer for a denaturing gel is Tris and glycine with detergent, and it is the buffer that sets up the stacking behaviour along with the gel and the sample buffer. It is usually made as a concentrate and diluted, and the one thing to watch is that the concentrate does not carry the detergent at a strength that precipitates in the cold. A buffer that has been standing in a cold room and looks cloudy is not ready to use.
An SDS running buffer is Tris, glycine and SDS
Named by its three components, which is the clearest way to keep it apart from the transfer buffer that shares two of them. The detergent is what keeps the protein unfolded and uniformly charged during the run; remove it and the separation stops being by size. It is also the reason a tank buffer can be reused a small number of times and then cannot: the ions deplete, the pH drifts, and the run slows and smears before anything looks obviously wrong.
Western blot wash buffer and antibody dilution buffer
The wash is a buffered saline with a little non-ionic detergent, and its job is to remove what is not bound without removing what is. The dilution buffer is usually the same saline with a blocking agent in it, so the antibody sits in the same chemistry it will be washed in. Keeping the two matched is worth more than any particular recipe, because a mismatch between them shows up as high background that looks like a blocking failure.
Questions people ask about western blot transfer buffer
Is the SDS page buffer the same as the transfer buffer?
No, and confusing them is a common way to lose a blot. Both contain Tris and glycine, but the tank buffer carries detergent to keep the protein unfolded, while the transfer buffer carries methanol to strip that detergent off so the membrane can bind it.
Can the western blot running buffer be reused?
A small number of times, if the runs are short. The ions deplete and the pH drifts, and the result is a slower, smeared run rather than an obvious failure, so discard it on a schedule rather than when it looks wrong.
Is a TBS buffer or a phosphate one better for the wash?
Tris-based for anything involving a phosphorylation-specific antibody, because phosphate in the wash competes with the epitope. For everything else either works, and consistency between the wash and the antibody dilution matters more than the choice.