Molecular weight markers for proteins

A protein marker is a lane of polypeptides with stated masses, run beside the samples so distance can be read as apparent size. The decision is which kind of marker can support the sentence you want in the figure legend. The photograph on this page is a gloved hand weighing white powder with a spatula inside an analytical balance draft shield. It is a weighing, not a gel and not a ladder. Weighing is how you measure a solid before you dissolve it. Choosing a marker is a different act, done from the mass you hope to see and from the gel system you will actually run. How a lane is read once the marker is there is covered in reading a protein gel. DNA ladders on an agarose gel are a different scale, explained in agarose gel electrophoresis for DNA.

Apparent mass is the thing the marker measures

SDS coats most proteins with negative charge roughly in proportion to length, and the polyacrylamide mesh sorts them. The position you record is an apparent mass under those conditions. It matches the sequence mass often enough to be useful, and it misses when the protein is heavily glycosylated, very basic, or incompletely unfolded. A reducing agent and heat are part of a denaturing prep. Leave them out and the marker, which was reduced, is no longer a fair neighbour to your lane.

UniProt is a public place to check the unmodified chain and known processing. Your construct wins if you added a tag the entry does not have. Write that expected apparent mass down before you shop for a ladder. A marker is then a bracket around that number, not a decoration.

The NIST Office of Weights and Measures is a public reminder that mass, as a measured quantity, has a traceable meaning on a balance. A coloured band on a gel is not that kind of measurement. It is a migration comparison. Keep the two ideas apart when the legend says "50 kilodaltons".

Three marker classes

Prestained markers carry a dye on the proteins so you can watch them during the run and see them on a transfer membrane afterward. They show orientation. They show that transfer happened across a size range. They are a poor sole sizing standard, because the dye shifts apparent mass, and the shift depends on the gel chemistry. A card that lists "apparent molecular weight" for a tris-glycine gel is not automatically true on a Bis-Tris gel at a different percentage. Use the prestain to know which end is which. Do the size call with a standard that was not shifted, or state clearly that you are reporting the prestained card's apparent values in that system only.

Unstained markers are ordinary proteins of stated mass. They appear when you stain the gel with the same stain you use on the samples, so the comparison is fair. On a western blot they may be invisible unless the detection sees them. Some unstained sets are modified so a later stain or a streptavidin reagent can find them. That modification can itself shift migration a little. Read whether the product is a sizing standard or a blot-trackable standard. If you need both jobs, run a prestained lane and an unstained lane, and say which one the legend uses for mass.

Fluorescent markers are the third class. They are visible on a fluorescent imager in a named channel and nearly useless on a bench transilluminator or a film that only records luminescence. They suit a fluorescent western or a fluorescent total-protein stain. They still need the bracket rule, and they still need the card's statement of whether the printed masses are dye-shifted. A channel mismatch looks like a blank ladder and sends people into a transfer panic. Check the imager first.

Loading dye is none of these. Bromophenol blue and its cousins mark the ion front so you know the run moved. They are not calibrated masses. An agarose DNA ladder is none of these either. Base pairs are not kilodaltons, and a nucleic-acid marker in a protein tank will not give you a protein scale.

A workflow with branches

Start from the mass and the percentage. A higher percentage resolves smaller proteins and can exclude a large one from the resolving gel, so the top band of a "wide range" marker may sit in the well and tell you nothing. A lower percentage does the opposite to a small protein, which then crowds the dye front. If the target is near the dye, choose a marker that still has a band below it, or change the percentage until one exists.

Branch if the prestained colours split or fade. The lot may have been warmed, or the gel system may not be the one on the card. Do not invent masses for pale bands. Run a known lysate you have sized before, and treat the marker as unverified until that lysate looks familiar.

Branch if the unstained marker and the sample stain differently. Some proteins take up Coomassie more readily than others. The marker still gives position. It does not give a mass quantity. Quantity belongs to a load you measured, which is where the balance in the photograph earns its place, together with a solution assay. Position and amount are two sentences.

On a blot, confirm the prestain survived transfer before you argue about the antibody. A ladder that transferred only at the small end means the large proteins, sample included, may still be in the gel. That check is part of western blot from gel to membrane.

Failure modes

Estimating past the end of the ladder is the failure this guide is for. A band below the last mark is "smaller than the last mark", not a calculated kilodalton. A band above the first mark is "larger than the first mark, or stuck". Say that.

Mixing a DNA ladder into the mental model is the next failure. An agarose image and an SDS-PAGE image do not share a ruler. A loading-dye front used as if it were 10 kilodaltons will move when the buffer system changes.

A marker lane that smiles cannot size a straight sample, and a straight marker cannot rescue a smiled sample. The shape problem is shared. Fix the run, then read.

Safety and the shipment

Marker proteins are usually a modest chemical hazard next to acrylamide monomer, and the vial still follows the sheet. The gel they run in may contain that monomer until it is polymerised. Casting stays under the institutional SOP. This page is not a clinical electrophoresis method.

Prestained and fluorescent markers often ask for cold storage. If a shipment arrives warm against that label, the dye bands are suspects until a known sample looks right. Cold-chain handoff, including how a frozen parcel is packed, is a separate operational note in dry ice packing for sample shipments. A warm afternoon in the loading bay is enough to matter. Write the condition on receipt next to the lot identity.

What the enquiry needs

EVRINTH can take a sourcing question. State the apparent mass you must bracket, the gel chemistry and percentage, whether you need prestained tracking, an unstained sizing standard, or a fluorescent channel, and whether the marker must be visible on a transfer membrane. The molecular biology catalogue is the list to point at, and the quote request carries the specification. The nucleic acid analysis pathway is relevant only when the same project also sizes nucleic acids, with a different ladder. Ask whether a quotation is possible. A range printed in a product title is not a promise that your protein falls inside it on your percentage.