1. Using a Microplate Reader for a variety of tasks
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!!