5x loading buffer western blot recipe, and what each component does

What each component does

1. SDS: the uniform negative charge. Sodium dodecyl sulphate coats the unfolded polypeptide with negative charge roughly in proportion to length, which is what makes migration a function of size. Too little and the separation is not by size; far too much and the front distorts.
2. Tris at pH 6.8: the stacking buffer. The sample buffer matches the stacking gel so the sample concentrates into a sharp band before it enters the resolving gel. A sample buffer at the wrong pH gives broad, smeared bands that look like a transfer problem.
3. Glycerol: density so the sample stays in the well. Glycerol makes the sample dense enough to layer under the running buffer. Concentrated stock is viscous, which is the practical reason a 5x buffer is harder to pipette accurately than a 2x one.
4. Bromophenol blue: a visible front. The dye tracks the ion front so you can see the run and stop it before the smallest proteins leave the gel. It is also the fastest way to spot a well that did not load.
5. A reducing agent, added fresh. A thiol reagent breaks disulphide bonds so the protein runs as a single chain. It oxidises in storage, which is why it is added to the aliquot on the day rather than kept in the stock. A stored complete buffer is the commonest reason a sample stops reducing properly.

Buying western blot buffers against making them, stripping buffer western blot included

A commercial buffer is consistent between lots, carries a safety data sheet and removes a weighing step from a busy day. Making it is cheaper and lets you adjust the SDS and reducing agent for an awkward sample. Most laboratories do both and use the commercial product as the reference when something stops working.

If you make it, prepare a large batch, aliquot it, and record the date. Small aliquots thawed once avoid both the freeze-thaw cycles and the temptation to top up an old tube.

Concentration and loading

A 5x stock leaves more room for sample volume than a 2x, which matters when the lysate is dilute. It is also viscous enough that pipetting accuracy suffers, so mix thoroughly and give the tip time to drain.

Load equal protein, not equal volume, and quantify the lysate before the buffer goes in. Detergent in the sample interferes with several protein assays, which is why quantification happens first.

Handling the reagents safely

SDS is a respiratory irritant as a powder and should be weighed in a hood or bought as a solution. Thiol reducing agents are odorous and toxic, and belong in a fume hood at every stage.

Every component needs its safety data sheet on file and its container labelled under the hazard communication rule, including buffers made in-house and decanted into a working bottle.

A running buffer western blot needs, and a stripping buffer for western blot reuse

The running buffer carries the current and the pH, and it is the one reagent people reuse until the separation degrades: the ions deplete from the cathode side first, so a second run is usually acceptable and a third is a gamble. Mix it from a concentrate you made or bought, and record which, because the concentrate's pH is what the run depends on.

Stripping to reprobe is a different chemistry and a different risk: a low pH or a detergent strip removes the antibody and some of the protein with it, so the second target is always measured on a membrane with less on it than the first. Where both targets matter equally, run two membranes rather than stripping one.

Common questions

How do I make a 5x western blot loading buffer?

Tris-HCl at pH 6.8, SDS, glycerol and bromophenol blue as a 5x stock, stored in aliquots at minus twenty. The reducing agent goes in on the day of use, because it oxidises in the freezer.

Should I always boil samples?

No. Ninety-five degrees for five minutes suits most soluble proteins. Multi-pass membrane proteins aggregate when boiled and often run better at thirty-seven or seventy degrees, which is a change worth testing before you conclude a protein is not expressed.

Reducing or non-reducing?

Reducing for most work. Non-reducing where the antibody recognises a conformational epitope or where you want to see the assembled complex, in which case the sample must not be heated hard either.

Why are my bands smiling or smeared?

Usually too much protein in the well, or a sample buffer whose pH or salt content has drifted. High salt samples distort the field locally, and diluting into fresh buffer rather than adding concentrated stock to a salty sample fixes most of it.

Is a Laemmli buffer western blot stock the same as this sample buffer?

Yes. A Laemmli buffer is the Tris, SDS, glycerol and bromophenol blue stock described above, named after the paper the discontinuous system comes from. The reducing agent is the only part that is not in the stored stock, and that is a storage decision rather than a difference of recipe.

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