Dynatech MR5000. Using a Microplate Reader for a variety of tasks. 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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Dynatech MR5000 Using a Microplate Reader for a variety of tasks
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
Applications • Kinetics • Agglutination • Tissue culture growth • Blood grouping • Complement fixation • MIC and bacterial ID assays • Advanced applications
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
Bradford Assay • Homogenization & Centrifugation • Preparation of Samples & Standards • Plate Loading and Bradford Reagant • Coomassie Brilliant Blue G-250 & H3PO4
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
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
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!!