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,Īnd 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 No conjugate is needed for fluorescent blot detection.Originally molecular weight markers were a mixture of easy-to-purify proteins of known molecular weight. Loading buffer: 30% (w/v) glycerol, 2% SDS, 62.5 mM Tris, pH 6.8, 50 mM DTT, 5 mM EDTA, 0.02% NaN 3, 0.01% bromophenol blue.įluorescent detection: prepare a 1:10 dilution of the WesternC Standard and load 5 µl per well. Fluorescent blot detection of prestained bands, with blue bands visualized in the red wavelength range and pink bands in the green wavelength range.Molecular weight sizing of proteins after blot development.Monitoring blot transfer efficiency with easy orientation via multicolor standards. These standards contain ten Strep-tagged recombinant proteins (10–250 kD), including three pink reference bands (25, 50, and 75 kD), as well as unstained versions of the Strep-tagged recombinant proteins for molecular weight sizing after blot development. Use Precision Plus Protein WesternC Standards as a combination standard for western blotting and fluorescence detection in SDS-PAGE. LC-MS innovation: Improve analytical accuracy Which microbioreactor modules are best for you Which solid support is right for your oligo synthesis?
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