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Choosing the appropriate sizing standard for protein electrophoresis.

Protein MW markers or protein ladders are mixtures of proteins of known molecular weight (MW) or molecular mass (kDa) that are used to assess the relative molecular sizes of proteins that have been separated by electrophoresis. Typically, molecular weight markers are run in the outer lanes of a gel for comparison to test samples that are run in the middle lanes.

A standard curve can be constructed from the distances migrated by each marker protein. Then the distance migrated by the sample proteins can be plotted and their molecular weights calculated by interpolation. More often, however, MW markers are used as a reference to help confirm the identity of proteins of interest whose sizes are already known.

What are Protein Ladders?

MW Marker vs. Protein Ladder

The name "MW marker" usually refers a mixture of native proteins whose sizes and mobilities are well characterized but do not correspond to whole numbers. "Protein ladder" generally refers to mixtures of undisclosed proteins (often prestained or labeled) whose sizes correspond to evenly spaced whole number values. Both types of product are considered protein standards for various purposes.

Unstained vs. Prestained MW Markers

Unstained MW markers are not visible during electrophoresis and must be stained with the rest of the gel to be seen. Prestained ladders are visible during electrophoresis, enabling the researcher to monitor the progress of separation for the range of protein sizes that are most relevant to the particular experiment. Most prestained markers and ladders also remain visible after gel staining or transfer to membrane for detection by Western blotting.

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Examples of unstained (left) and prestained (right) protein ladders. To visualize an unstained ladder (left), the proteins in the gel or blot must be stained after electrophoresis. Prestained ladders are visible during and immediately following electrophoresis, as well as after total protein staining.

Typical blue prestained markers are bound non-covalently with coomassie blue dye, the same dye often used for gel staining. This sort of prestaining is not an exact process, and prestained markers do not provide the same size precision as unstained proteins. The apparent molecular weight of a prestained protein is usually greater than the unstained protein, and lot-to-lot variation is normal.

Many commercial prestained protein markers are multicolored, including many of our Thermo Scientific Protein Ladders. In addition to blue-stained bands, these ladders include selected bands that are green, orange or red. These alternative colors in the ladder provide unambiguous reference points for identifying which band corresponds to which molecular weight, even if some bands were run off the bottom of the gel.

Selection table for Thermo Scientific Pierce MW markers and ladders

Bars in the following info graphic indicate the ranges of each product listed at the left side. Diamonds indicate the location and color of protein bands in each protein ladder. The names and chart-data are hyperlinked to product pages.

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MW Markers for Chemiluminescent Western Blotting

Thermo Scientific SuperSignal Molecular Weight Protein Ladder and SuperSignal Enhanced Molecular Weight Protein Ladder contain ready-to-use stabilized mixtures of eight recombinant proteins ranging in size from 20 to 150kDa. These recombinant proteins contain a functional polypeptide sequence that binds antibodies used in the Western blot. Consequently, the protein markers can be visualized either using appropriate substrates for enzyme-labeled antibodies or via fluorescent dye-labeled antibodies. The MW marker mixes also contain a special pink tracking dye to monitor the electrophoresis run and membrane transfer without the need for an additional colored markers. The standards are compatible with nitrocellulose and PVDF membranes, multiple blocking buffers and many other detection methods.

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|Thermo Scientific SuperSignal Protein MW Ladder.| Proteins in this MW marker can be visualized by chemiluminescent Western blot (A), by gel stains based on coomassie blue dye (B) or silver (C).

MW Marker for Near-Infrared Fluorescence Imagers

The Thermo Scientific PageRuler Prestained NIR Protein Ladder is a mixture of 10 proteins (11 to 250KDa) that are blue-stained and fluor-labeled for near-IR fluorescent visualization and protein sizing following electrophoresis. The protein MW markers in this ladder resolve into sharp bands when analyzed by SDS-PAGE and are labeled with a fluorescent dye for visualization with instruments equipped for detection of near-infrared (NIR) fluorescence. These include Typhoon* Imagers and the LI-COR Odyssey* Infrared Imaging System.

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|SDS-PAGE band profile of the Thermo Scientific PageRuler Prestained NIR Protein Ladder.| Images represent the same 4-20% Tris-glycine gel (SDS-PAGE) visualized directly with the unaided eye (left) and via near-infrared fluorescence lasers with the Typhoon* Imager (GE Biosciences). Similar fluorescence imaging results are obtained with the LI-COR Odyssey* System.