Dynatech%20MR5000 - PowerPoint PPT Presentation
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Dynatech%20MR5000
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... more involved than Bradford Enzyme-Linked Immunosorbent Assay Buffers 1 and 2 antibodies Color Detection ... Antibody ELISA Bradford data ... – PowerPoint PPT presentation
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Title: Dynatech%20MR5000
1
Dynatech MR5000
- Using a Microplate Reader for a variety of tasks
2
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
3
Applications
- Kinetics
- Agglutination
- Tissue culture growth
- Blood grouping
- Complement fixation
- MIC and bacterial ID assays
- Advanced applications
4
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
5
Bradford Assay
- Homogenization Centrifugation
- Preparation of Samples Standards
- Plate Loading and Bradford Reagant
- Coomassie Brilliant Blue G-250 H3PO4
6
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
7
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
8
HSP 70
9
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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