Protein ladder selection for western blot: which kind, and how to read it

Choosing and using the ladder

1. Prestained, for watching the run and the transfer. Dye-coupled proteins are visible in the gel and on the membrane, so you can see the separation develop and confirm the transfer before any antibody is used. The dye coupling shifts mobility, which is why the stated weights are approximate.
2. Unstained, where the size estimate has to be as good as it can be. Without dye the protein runs closer to its true mobility, so sizing is better. It is invisible until stained, which makes it useless for monitoring and means the lane has to be stained or the ladder detected separately.
3. Dual colour, to anchor the eye. Reference bands in a contrasting colour at known positions let you read the ladder at a glance without counting from the top. On a blot photographed in a hurry that is worth more than it sounds.
4. Match the range to the target, not to the catalogue. A ladder spanning ten to two hundred and fifty kilodaltons is fine for a forty kilodalton protein and useless for resolving two bands ten kilodaltons apart at the bottom of the gel. Pick a ladder whose bands bracket the target closely.
5. Handle it like a reagent that degrades. Ladders tolerate a limited number of freeze-thaw cycles before bands fade and smear. Aliquot on arrival, and do not heat a prestained ladder harder than the instructions say: overheating is the usual cause of a ladder that suddenly runs as a smear.

Reading a size off a gel without overclaiming

Migration is a function of the detergent-coated chain, so anything that changes the coating changes the apparent size. Heavily glycosylated proteins run larger, some acidic proteins run smaller, and phosphorylation shifts bands by more than the modification's mass.

State the number as an apparent molecular weight and say what system produced it. A band described as running at its expected apparent weight on a given gel is an honest claim; a band described as being a particular mass is usually not.

Ladders on the membrane, and the protein gel ladder that survives transfer

A prestained ladder transfers with the sample and marks the membrane, which is what lets you cut a blot at the right height for two different antibodies. Marking the positions in pen immediately after transfer saves an argument later, because some dyes fade during blocking.

If the imaging system reads chemiluminescence only, the ladder will not appear in the image at all. Capture a colour photograph of the membrane before detection and overlay the two, or use a ladder the imager can see.

When the ladder is the problem

Fading, smearing and missing bands are almost always storage: repeated freeze-thaw, a fridge-stored vial, or heating a prestained ladder to boiling. A fresh aliquot settles it immediately.

A ladder that runs at an angle while the samples run straight indicates uneven gel polymerisation or a well that was damaged loading, not a problem with the reagent.

A low molecular weight ladder, and reading the small end

A low range ladder covers roughly two to forty kilodaltons and exists because a standard broad-range set has few bands where small proteins and peptides run. Reading the small end also depends on the gel: a Tris-tricine or a high percentage gel with a suitable buffer resolves what a standard glycine system pushes into the dye front. Prestained ladders are convenient and their apparent masses shift with the gel system, so an accurate size is estimated against an unstained standard run in the same gel and transferred alongside the sample.

A dual color protein ladder and what the second colour is for

A dual color protein ladder has two or more bands in a contrasting colour at defined masses, which makes orientation and mass estimation possible at a glance during a transfer, and the coloured bands are visible on the membrane so transfer can be confirmed before detection. For fluorescent detection a ladder in the imaging channel is what appears beside the sample bands.

A tff membrane and the cut off that is chosen

A tff membrane is specified by its molecular weight cut off, its material and its area, and the cut off is chosen several fold below the product's mass because retention is a distribution rather than a wall. Material decides binding losses and cleanability. Transmembrane pressure and crossflow are the operating parameters that decide flux and whether the membrane fouls.

western blot molecular weight markers and what they can tell you

western blot molecular weight markers estimate mass, and the estimate is only as good as the separation: glycosylation, phosphorylation and detergent all move a band, so an apparent mass that disagrees with a calculated one is usually real rather than an error. A prestained ladder confirms transfer while a detectable ladder appears on the film beside the sample.

Common questions

Prestained protein ladder or unstained protein ladder?

Prestained to monitor the run and the transfer, unstained when the molecular weight estimate matters. Many laboratories run both, on opposite sides of the gel.

Why do the ladder weights not match the literature?

Because apparent molecular weight depends on the gel system, and prestained ladders are shifted further by the dye. The values printed on the vial are calibrated for a specific gel chemistry, and they change if you change it.

Can I use a western blot protein ladder for quantitation?

Only if it is sold as a quantified ladder with a stated mass per band. Ordinary ladders are for position, not for amount, and treating band intensity as a standard curve will mislead you.

How much ladder should I load?

The volume on the datasheet, and no more. Overloading produces a bright smear that bleeds into the neighbouring lane, which is a recurring cause of a phantom band at the edge of the first sample.

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