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Published byLeon Aldous Horton Modified over 9 years ago
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Using a Microplate Reader for a variety of tasks
Dynatech MR5000 Using a Microplate Reader for a variety of tasks
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Features of the Dynatech MR5000
Microprocessor controlled photometer 96-well microplates MICROELISA MICROTITER 5 different filters 405, 450, 490, 550, 595 (nm) Single and dual wavelengths eliminates some error
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Applications Kinetics Agglutination Tissue culture growth
Blood grouping Complement fixation MIC and bacterial ID assays Advanced applications
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What I do with the MR5000 Bradford Assay
total protein concentration of a sample quick and easy ELISA - more involved than Bradford Enzyme-Linked Immunosorbent Assay Buffers 1 and 2 antibodies Color Detection
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Bradford Assay Homogenization & Centrifugation
Preparation of Samples & Standards Plate Loading and Bradford Reagant Coomassie Brilliant Blue G-250 & H3PO4
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What the Bradford tells us
Total protein concentration for each sample Why is this important? To run ELISA need same amount of protein in each sample – for my work 2000 ng For ELISA use a particular volume for each sample (determined by Bradford readings) FEB BRADFORDS completed.xls
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Monoclonal Antibody ELISA
Bradford data Standards Reagants Binding Buffer (10mM Na2CO3/NaHCO3) Blocking Solution – PBST (PBS + Tween 20) PBST-BSA Antibodies – Primary Secondary Color Detection Solution Diethanolamine R-nitrophenylphosphate tablets DAS-ELISA
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HSP 70
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Summary So, we have: Determined the concentration of protein in a sample (Bradford 595 nm) Quantified the concentration of HSP 70 in equal volumes of protein (ELISA 405 nm) Had a lot of fun!!
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