Dynatech%20MR5000

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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!!