The reagents that decide what reaches the gel: why a western blot lysis buffer, and the lysis buffer for western blot and lysis buffer western blot phrasings beside it, has to be chosen for the compartment the target lives in rather than as a default recipe, what an sds page buffer, an sds running buffer and an sds-page running buffer do differently and why the running buffer is not the sample buffer, where bsa for western blot blocking, western blot bsa and the bsa western blot habit beat milk and where they do not, what a nitrocellulose membrane for western blot work, or a nitrocellulose membrane western blot generally, gives up against a polyvinylidene fluoride one and where a regenerated cellulose membrane belongs to filtration instead, and what western blot analysis software has to preserve about the raw image before any number it produces can be defended

Choosing the reagents

1. Choose lysis for the compartment. A mild buffer leaves membrane, nuclear and cytoskeletal proteins in the pellet. A strong denaturing buffer solubilises almost everything and destroys native interactions. Decide which your target and your question need, and blot the pellet once to check.
2. Keep inhibitors in the buffer, not beside it. Protease and phosphatase inhibitors have to be present before the cells meet the buffer, made fresh, and dated. Adding them afterwards is the commonest reason a signal is lost.
3. Do not confuse sample buffer with running buffer. The sample buffer denatures, reduces and loads; the running buffer carries the current and sets the ion front. Substituting one for the other is a mistake that produces a characteristic mess.
4. Match the membrane to the protein and the detection. Nitrocellulose gives low background and is fragile; polyvinylidene fluoride binds more and tolerates stripping and reprobing. For small proteins the pore size matters more than the material.
5. Choose the blocker against the target class. Milk based blockers carry phosphoproteins and defeat phosphospecific detection. Protein free and albumin based blockers suit those cases. A blocker change is a method change.
6. Keep the raw image and the settings. Any number from a blot depends on the exposure and the analysis regions. Software that exports only an adjusted image makes the number indefensible.

The buffer decides the experiment

Which proteins appear on a blot is decided by the lysis buffer, not by the antibody. A laboratory that runs one buffer for everything is systematically blind to whole classes of target and usually does not know it.

Keep two or three documented buffers for different compartments, and record which was used with every result.

Defensible numbers need raw data

A densitometry value depends on the exposure chosen, the region drawn and the background subtraction method. Without the raw image and the settings, none of that can be re-examined.

Archive the unadjusted image, the acquisition settings and the analysis file with the figure. It is what turns a blot into evidence.

Common questions

Why is my target missing from the lysate?

It is probably in the pellet. Membrane, nuclear and cytoskeletal proteins need stronger extraction than a mild buffer provides. Blot the pellet once and the question is settled for that cell type.

Milk or albumin blocking?

Albumin or a protein free blocker for phosphospecific detection, because milk contains phosphoproteins. Milk is cheaper and effective otherwise. The target decides.

Nitrocellulose or polyvinylidene fluoride?

Nitrocellulose for low background and easy handling; the fluoride membrane for higher binding capacity, durability and reprobing. Pore size matters more than material for very small proteins.

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