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Short answer: a protein marker, also called a protein ladder or molecular weight standard, is a lane of proteins of known sizes run alongside your samples on a gel so you can track the run, follow transfer to a membrane, and estimate the size of the bands you care about. Prestained markers are colored and visible during the run; unstained markers are visualized after staining and are better for precise sizing.
Definition and scope
A protein marker is a mixture of proteins whose molecular weights are known, loaded in a lane of an SDS-PAGE gel (or another gel format) next to the samples. After electrophoresis, the marker bands form a reference scale. Manufacturers describe the same product class with different words: marker, ladder, standard. This page treats them as one thing. For the gel itself, see SDS-PAGE protein gel electrophoresis; for what happens after the gel, see western blot protocol basics.
Western blot ladder and protein ladder for western blot
A “western blot ladder” is simply a protein marker chosen to be useful on a blot. Two properties matter there. Prestained markers are colored so you can watch the proteins separate and see them move onto the membrane; Thermo Fisher Scientific describes its prestained markers as compatible with western blotting. Some markers are also built for detection: they are recombinant proteins fused to an IgG-binding site, so they bind the primary or secondary antibody and appear on the blot as a built-in positive control.
Prestained protein ladder
A prestained ladder carries covalently attached dye. The dye is what lets you see the bands without staining, but Thermo Fisher notes that unstained markers are the ones “not labeled with dyes that can alter the apparent molecular weight”. In other words, the dye is a trade-off: visibility in exchange for some uncertainty in size.
How a marker is used on a gel and a blot
The marker occupies its own lane. During the run it shows whether separation is proceeding evenly across the gel. After transfer, a prestained marker shows on the membrane whether proteins moved across. When the blot is probed, the marker bands are your size scale for judging where a signal sits; the guide on how to interpret western blot results covers reading band size, extra bands and controls.
Loading volume is product-specific. As one example, the manufacturer’s instructions for the Thermo Fisher iBright Prestained Protein Ladder give 1 to 3 µL per well for a 12- or 20-well mini gel, 2 µL for a 26-well gel and 2 to 4 µL for a midi gel, and state that the ladder is ready to use, with no heating, dilution or reducing agent, and should not be boiled. Always follow the instructions for the product in your hands rather than carrying these numbers across brands.
Types of protein marker
The table maps the purpose of the lane to the class of marker, using the Thermo Fisher protein standards overview. Product names are examples from that page, not recommendations.
| What the lane is for | Marker class | Example from the source |
|---|---|---|
| Watch separation and transfer, approximate size | Prestained | PageRuler Plus, 10 to 250 kDa; HiMark, 30 to 460 kDa (for Tris-Acetate gels) |
| Precise sizing without dye effects | Unstained, visualized after staining (for example Coomassie) | PageRuler Unstained, 10 to 200 kDa; HiMark Unstained, 40 to 500 kDa |
| Positive control on a western blot | IgG-binding recombinant proteins | MagicMark XP (IgG binding on all bands); iBright (two such bands) |
| Small proteins | Low-range prestained | Spectra Multicolor Low Range, 1.7 to 40 kDa (for Novex Tricine gels) |
| Isoelectric point | IEF marker | IEF Marker 3-10 |
| Native gels | Native marker | NativeMark |
| His-tagged, phospho- or glycoproteins | Specialty markers | BenchMark His-tagged; Peppermint Stick (phosphoproteins); CandyCane (glycoproteins) |
For isoelectric focusing, which separates by charge rather than size, see isoelectric focusing, pI separation, ampholytes and IPG strips.
Where it matters for labs and supply decisions
- Match the marker to the gel chemistry. Most markers on the Thermo Fisher page are listed for all SDS-PAGE gels, but some are gel-specific: HiMark and PageRuler Unstained High Range for NuPAGE Tris-Acetate gels, Spectra Multicolor Low Range for Novex Tricine gels, NativeMark for native PAGE. The source advises matching the ladder to the gel chemistry (Tris-Glycine, Bis-Tris, Tricine) and checking each product’s specifications.
- Check the labeled range against your target. Ranges differ widely, from 1.7 kDa at the low end of one product to 500 kDa at the high end of another, so a marker has to bracket the size you expect.
- Check the labeling and storage. The iBright Prestained Protein Ladder is labeled “For Research Use Only” and is stored at -20 °C (unused ladder returned to -20 °C can be kept up to one year), per its product information sheet (Pub. MAN0016682 Rev. E.00, 20 July 2017).
What prestained markers can and cannot tell you
This is where the manufacturer’s own instructions are more careful than most overviews. The iBright product information states:
- The mobility of prestained proteins can vary between SDS-PAGE buffer systems. They are suitable for approximate molecular weight determination when calibrated against unstained standards in the same system.
- The apparent molecular weight variance of its 10 prestained proteins is about 5%.
- In low-percentage gels (below 10%), the low molecular weight proteins in the ladder may migrate with the dye front.
- Large proteins (above 100 kDa) may require longer transfer times or higher transfer voltages.
- With fluorescent detection, a low molecular weight band from free dye may be seen.
- Its two unstained bands (30 and 80 kDa) need Coomassie or a reversible membrane stain to be seen.
The practical reading: a prestained band is a guide to size, and the band you see is not the same as a calibrated size. If the size call matters, calibrate against an unstained standard run in the same system.
Common misunderstandings
“A prestained band gives the exact size.”
It does not. Dye alters mobility, buffer systems change it further, and one manufacturer quotes about 5% apparent variance for its prestained proteins.
“Every marker works on every gel.”
Most listed products suit SDS-PAGE gels broadly, but several are specific to a gel chemistry or to native PAGE. Check the product specifications.
“Heat it like a sample.”
Not necessarily. The iBright ladder is ready to use and the instructions say not to boil it. Other products may differ, so read the instructions for the specific marker.
“The marker is a quantity standard.”
The sources describe markers as size references, run-and-transfer trackers and, for IgG-binding types, a positive control for antibody detection. They do not describe them as a way to quantify a sample protein.
FAQ
What is a protein marker?
A mixture of proteins of known molecular weights run in a lane next to samples, used to track the run and transfer and to estimate band sizes. It is also called a protein ladder or molecular weight standard.
What is a prestained protein ladder?
A ladder whose proteins carry dye, so the bands are visible during the run and on the membrane after transfer. The dye can alter apparent molecular weight, so prestained ladders suit approximate sizing.
Which protein ladder works for a western blot?
Prestained ladders are described as compatible with western blotting, and IgG-binding ladders add a positive control that lights up with the antibody. The right choice also depends on your gel chemistry and the size range of your target.
Can I use a western blot ladder for a native gel?
Not by default. The source lists a separate native-gel marker, NativeMark, for native PAGE; check the specifications of any marker before using it outside SDS-PAGE.
Sources
- Thermo Fisher Scientific, Protein ladders and standards for SDS-PAGE, western blots, and IEF. Checked 2026-10-29.
- Thermo Fisher Scientific, iBright Prestained Protein Ladder Product Information, Pub. MAN0016682 Rev. E.00 (20 July 2017). Checked 2026-10-29.
Product names appear as examples taken from these pages. They are not recommendations, and nothing here is a statement about price, availability or suitability for a particular experiment.
