Dynatech MR 5000 Using a Microplate Reader for

Dynatech MR 5000 Using a Microplate Reader for a variety of tasks

Features of the Dynatech MR 5000 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 MR 5000 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 & H 3 PO 4

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 1. 2. 3. Bradford data Standards Reagants • • • 4. Antibodies – • • 5. Binding Buffer (10 m. M Na 2 CO 3/Na. HCO 3) Blocking Solution – PBST (PBS + Tween 20) PBST-BSA Primary Secondary Color Detection Solution 1. Diethanolamine R-nitrophenylphosphate tablets DAS-ELISA

HSP 70

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!!