PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Electrophoretic Profiles of Different Protein Molecular Weight Markers by Ghanshyam D. Heda, Ph.D. Department of Veterans Affairs V.A. Medical Center Memphis, TN INTRODUCTION In protein molecular weight markers, precision, accuracy, and reproducibility of electrophoretic separation are essential for protein analysis. A variety of protein molecular weight markers are commercially available from different vendors. These markers are available as prestained, unstained, and biochemically altered, such as biotinylated, for example. The advantage of prestained markers is that the separation of protein bands can be visualized during the electrophoretic run. Unstained markers require staining and de-staining for visualization. Electrophoretic separation of any given set of protein molecular weight markers depends on the concentration of polyacrylamide gel. Gradient gels are ideal for visualization of a wide range of proteins from low molecular weight (as low as 10 kDa) to high molecular weight (as high as 250 kDa). The aim of this article is to analyze the protein profiles of the following sets of molecular weight markers: stained and unstained, low and high, and biotinylated and un-biotinylated molecular weight markers. MATERIALS AND METHODS A 7.5 µl of each stained and unstained marker listed in the chart was applied directly without any alteration or addition to a 4-15% linear SDS-PAGE gel (BioRad Laboratories) and electrophoresed at 25 mA constant current for ~ 2.5 hours or until the dye-front reached to the bottom edge of the gel. ECL DualVue Western Blotting Marker was purchased from GE Healthcare (formerly Amersham Biosciences), and Benchmark™ was purchased from Invitrogen. All other markers were purchased from BioRad Laboratories. The gel was digitized before staining (Figure 1) and again after staining with Simply Blue™ safe stain (Invitrogen) and de-stained with water according to the manufacturer’s protocol (Figure 2). RESULTS Any time a molecular weight marker is compared with another marker, regardless of its range (i.e., wide range or high molecular weight range) some variation – from slight to large – in the protein profile was noticed. For example, the 32.1 kDa mark of BR161-0324 runs parallel to the 26 kDa mark of Benchmark™. Further, variation can occur even among products obtained from the same vendor. For example, the129 kDa mark of BioRad’s BR161-0324 runs parallel to the 116.5 kDa mark of another BioRad product, BR161-0309. However, biochemical alteration of a marker such as pre-staining (161-0363 versus 161-0373) and/or biotinylation (161-0303 versus 161-0311) had no effect on the protein profile. Lot-to-lot variation of the same product [e.g., BR161-0309 (See Chart, re: Lot 1) to 161-0309 (See Chart, re: Lot 2)] was minimal.  Chart Molecular weight sizes of all protein bands shown in Figures 1 and 2 were provided the by manufacturer.  Figures 1 and 2 DISCUSSION Careful selection of protein molecular weight markers is essential and must be made based on concentration of SDS-PAGE. For low-concentration gels, high-range molecular weight markers are ideal. Wide-range molecular weight markers, on the other hand, are suitable for gradient gels. It is important to confirm the molecular weight size of a protein ladder each time a new lot is obtained. Using the same product for the entire duration of the project may reduce errors since variability exists between products. This is true even if they are obtained from the same vendor. However, for accurate analysis of proteins, a comparative electrophoretic run of more than one product from the same and/or different vendors is recommended. To read more about the author, click here to read his Member Spotlight Article. ### << Previous Next >> [ View All Perspectives ] |
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