Western blot lysis buffer and the reagents that decide what reaches the gel

Choosing the reagents, including a blocking buffer western blot protocols specify

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 lysis and sample buffer decides the experiment: western blot sample buffer choices

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.

Western blotting reagents, and which of them you should make

Western blotting reagents divide into the ones worth buying and the ones worth making. Buying is sensible where consistency decides the result: the lysis buffer with its protease and phosphatase inhibitors, the blocking reagent, the transfer stack and the detection substrate, whose sensitivity drifts as it ages. Making is sensible for the simple salt solutions, running and transfer buffer, wash buffer, and for the sample buffer if you need to change the reducing agent. Cost the bought list per blot rather than per bottle, because the substrate and the antibodies dominate it.

The blocking solution western blot protocols ask for, and what it competes with

A blocking solution coats the membrane so the antibodies bind their target rather than the nitrocellulose, and the choice interacts with the probe. Milk is cheap and effective and contains phosphoproteins and biotin, so it is wrong for a phospho-specific antibody or a streptavidin detection. Bovine serum albumin avoids both and blocks less completely, so backgrounds rise. Fish gelatin and commercial protein-free blockers exist for the same reason. Block in the same detergent concentration the antibody dilution uses, and change one variable at a time when a blot comes up dirty.

sds page running buffer 10x, and making it properly

A ten times Tris-glycine-SDS stock is Tris base, glycine and SDS in water, and two habits keep it working. Do not adjust the pH of the concentrate: the buffer's pH arises from the Tris and glycine ratio when diluted, and adding acid or base to the stock changes the chemistry rather than correcting it. And dilute with good water to one times before use, since SDS precipitates in a cold concentrate and needs warming to redissolve. Reusing running buffer costs resolution as its ions deplete, so a second run is a compromise rather than a saving.

Common questions

Why is my target missing from the RIPA buffer western blot 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 in TBS western blot buffers?

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

Which western blot membrane: nitrocellulose membrane for western blot 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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