Molecular weight (within limits) is irrelevant. An IEF protein ladder contains native proteins with a range of isoelectric points. This is often followed by SDS-PAGE in the second dimension (2-D SDS-PAGE). IEF (Isoelectric focusing) and 2-D SDS PAGE Ladders: In IEF, proteins migrate through an immobilized pH gradient until they get to their isoelectric point (pI) where they have no charge and cease migrating. See the product overview pages for specific details.īoth fluorescently labeled proteins and chemiluminescence can be detected and analyzed with instruments such as the ChemiDoc™ or Gel Doc™ imaging systems. StrepTactin-HRP and StrepTactin-AP are available.įluorescent properties: Many of the stained recombinant proteins are excited by wavelengths used in fluorescent detection systems. High-affinity binding of StrepTactin, a genetically modified form of streptavidin, gives convenient and simultaneous detection of both proteins and standards on western blots using either colorimetric or chemiluminescence methods. All prestained recombinant ladders include three high-intensity reference bands (25, 50, and 75 kD).įor size determination on western blots, depending on the visualization method being used, the protein ladder can be unstained, prestained, or with a Strep-tag sequence. With an unstained protein ladder, only the bromophenol blue at the dye front is used to monitor the progress of the gel and blotting efficiency is not readily determined. Prestains and Tags: Proteins within a ladder may be stained all one color, have two or more bands stained different colors to make sure that the molecular weights within the ladder are easily identified, or have a multicolored pattern like Kaleidoscope™ protein standards.Īn advantage of using a prestained protein ladder is that the migration of the size range of the polypeptides of interest can be monitored during electrophoresis. Every recombinant protein ladder is broad range. Unstained natural protein standards also come in a polypeptide range (1.4–26.6 kD). Molecular Weight Range: Natural protein standards come in high molecular weight range, low molecular weight range, and broad molecular weight range. The recombinant proteins used in protein ladders have been engineered to have tight bands, as well as specific traits such as evenly spaced molecular weights, individual proteins stained with different colors for easy identification, or affinity tags for detection on western blots. ![]() Though there are many laboratories that continue to use natural protein ladders, most now use recombinant standards. The broad, less distinct, bands make them less attractive for molecular weight estimation. Prestained natural proteins may produce broader bands than recombinant proteins, particularly when the proteins are prestained with more than one color. Recombinant Proteins: There is variability for prestained naturally occurring proteins, in the amount and location of dye that binds to each protein. These marker proteins were unstained, and were generally visualized on SDS-PAGE gels by staining the gel with Coomassie Brilliant Blue R250, or after western transfer by a stain such as Ponceau S. Membrane-compatible – colored bands transfer to membranes for Western blottingĭye-stained proteins in 62.5mM Tris-H 3PO 4 (pH 7.5 at 25☌), 1mM EDTA, 2% SDS, 10mM DTT, 1mM NaN 3 and 33% glycerol.Originally molecular weight markers were a mixture of easy-to-purify proteins of known molecular weight. Quality tested – each lot evaluated by SDS-PAGE and Western blottingīright reference bands – orange at 70 and 25kDa, and green at 10kDa Sharp bands – color-coded bands of similar intesity for easy visualization Ready-to-use – supplied in a loading buffer for direct loading on gels no need to boil Size range – nine proteins spanning 10 to 250kDa PageRuler Plus Prestained Protein Ladder is ready to use: no heating, further dilution or addition of a reducing agent is required before loading onto a gel. A blue chromophore is bound to all proteins, except proteins of two reference bands of 70kDa and 25kDa that are colored with an orange dye and one green reference band of 10kDa. This prestained protein MW marker is designed for monitoring the progress of SDS-polyacrylamide gel electrophoresis, for assessing transfer efficiency onto PVDF, nylon and nitrocellulose membranes, and for estimating the approximate size of separated proteins that have been made visible with gel stains or Western blot detection reagents. ![]() Thermo Scientific™ PageRuler Plus Prestained Protein Ladder is a mixture of nine (9) blue-, orange- and green-stained proteins (10 to 250kDa) for use as size standards in protein electrophoresis (SDS-PAGE) and Western blotting.
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