Therefore, it is important to allow the reaction to proceed until color development is satisfactory and then stop the reaction. In contrast to chemiluminescent western blotting, the colored precipitate formed by chromogenic substrates cannot be easily stripped off to facilitate re-probing procedures. Similar to developing film, the blot is incubated in substrate until the desired amount of development is achieved. The appropriate substrate choice depends on the enzyme label and desired sensitivity. Chromogenic blotting substrates are available in a variety of specifications and formats, producing a range of colored precipitates. The resulting colored band or spot requires no special equipment for processing or visualizing. Alkaline phosphatase- AP or horseradish peroxidase-HRP), they are converted to insoluble, colored products that precipitate onto the membrane. When these substrates come in contact with the appropriate enzyme (e.g. The ChemiDoc MP Imaging System is an instrument for imaging and analyzing gels and western blots. Best application for high abundant proteins and when imaging or film processing instrumentation is not availableĬhromogenic or precipitating substrates have been used widely for many years and offer the simplest and most cost-effective method of western blot detection.Stripping and reprobing of blot not possible.Longer exposure times can produce high background because of the small amount of excitation light passing through emission filters Right Figure and Table Below: Comparison of the limit of detection for RNase A on Jess using chemiluminescence immunoassay and Stellar NIR (red bands) and Stellar IR (green bands) detection modules with a leading competitor Western blot fluorescence imaging system.Care is needed to avoid fluorescence contamination.Long exposure times possible, as no excitation light source required to capture signal.Stripping and reprobing of blot possible.Multiplexing with an internal control makes normalization simpler Single-channel detection makes normalization challenging Imaging instruments with appropriate filters or lasers Possible variation between blots, can be mitigated by using high duration substrates Good, may require higher concentration of secondary antibody Simultaneous detection of multiple proteins on the same blot using a variety of available fluorescent dyes and conjugated 2° antibodiesĭirect visualization of your target protein using color detection reagentsĮxcellent, with a wide variety of substrates available Enhanced chemiluminescent (ECL) HRP and AP substrates providing pictogram to femtogram level detection
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