What is the most sensitive staining method without using Silver Stain? Colloidal CBB G250 can stain with a detection limit of 1ng. But can any other non silver staining method go beyond?
Silver Staining is highly sensitive and go upto Femto Molar Concentration. However, its not MS Compatible and requires special modifications for MS that lowers the sensitivity. You can use Sypro-Ruby or Fluoro Tect or Deep Purple. These dyes offer high sensitivity (nanogram level) and are compatible with MS. Deep Purple, for example, can stain proteins in the sub-nanogram range.
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Thanks Biographix. Would these staining methods also require a special detection system for Fluorescent dyes or a simple UV filter and detector in regular Gel Doc work? We are currently using Colloidal CBB G250 and combined with high temperature staining it is staining around 1ng protein.
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To detect fluorescently stained proteins, you typically need a fluorescence imaging system that includes: Appropriate light sources (e.g., LED or Xenon lamps) that emit the specific wavelength required to excite the fluorescent dye. Filters to separate the excitation and emission wavelengths, ensuring that only the emitted fluorescence from the protein stain is detected. The regular gel docs are equipped with UV typically for DNA visualization that is based on intercalation of EtBr. These aren’t sufficient for Protein detection.
How about Zinc Imidazole Negative staining method? That is more sensitive that traditional Coomassie Brilliant Blue but is MS Compatible at the same time. Have you tried that?
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Zinc Imidazole staining method is simple but if you’re not used to Negative staining, it will require conversion using your Gel Documentation system by converting background into Score and invert the picture. Densitometry may not be accurate but yes, it is comaptible with MS because your protein is still unstained.
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