Topics to be covered Principles of immunological detection methods

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Presentation transcript:

Topics to be covered Principles of immunological detection methods Formats of ELISA assays, direct, sandwich Immunoblotting Substrates for detection Chromogenic, fluorogenic substrate, fluorescence labeled detection molecules Other methods, RIA and immunofluorescence microscopy

Sandwich ELISA and other formats

SDS-PAGE & Immunoblotting X Substrate reaction produces a chemiluminescent signal HRP: horse raddish peroxidase

Substrates for detection Substrate reaction produces a chemiluminescent signal HRP: horse raddish peroxidase

SDS-PAGE & Immunoblotting Cell extracts and affinity purified samples will be analyzed using SDS-PAGE and immunoblotting A putative result: Reducing conditions 130 kDa α -IgG-HRP 100 kDa 70 kDa Secretion signal -hc 55 kDa hc 35 kDa 25 kDa EndoH - + - + - + - + - + - + IgG 10 ng HyHEL -Fv-Fc-fusion Her2 IgG HyHEL IgG

Other immunological detection methods Immunofluorescence RIA See also: https://www.thermofisher.com/fi/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods.html http://www.bio-rad.com/en-fi/life-science-research