Transcript Document

Protein Structure
and Analysis
Activity 7.1: Protein Quantitation
Using the Bradford Assay
Activity 7.1: Protein Quantitation Using
the Bradford Assay
 Research question:
– How much protein is present in milk?
 Objectives:
– Perform a Bradford assay on two milk samples
– Perform a Bradford assay on a series of standards of known protein
concentration
– Use a spectrophotometer to measure the absorbance of Bradford
assay samples
– Generate a standard curve of protein concentration against the
absorbance at 595 nm
– Determine the protein concentration in the milk samples
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Safety Reminders
 Follow all laboratory safety procedures
– Read and become familiar with MSDS for the
Bradford reagent
– Wear appropriate PPE
– Bradford reagent can stain the skin and clothes
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Skills to Master
 Perform a Bradford assay
 Use a spectrophotometer (Activity 5.4)
 Generate a standard curve using absorbance
values
 Determine experimental values using a standard
curve
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Tips
 Mix samples thoroughly with the Bradford reagent
– Pipet up and down or cover with Parafilm and invert to mix
 Samples should be incubated at room temperature
for at least 5 min after mixing with the Bradford
reagent
 Orient cuvettes correctly in the spectrophotometer so
that light passes through the sample
 Do not handle the cuvette where light passes since
fingerprints can interfere with the reading
 If a spectrophotometer is not available, visually
compare the samples to the protein standards
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Student Workstation Materials
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Determining Protein Concentration
 Play video: Bradford Assay
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Protocol Highlights/Tips
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Make sure samples are mixed
thoroughly
Touch the cuvettes only at the top
Make sure the cuvettes are oriented
properly when placing them into the
spectrophotometer
Ensure that the samples and
controls have incubated 5 minutes
after mixing with the Bradford
reagent before analysis
Compare unknown samples to
controls by eye before using the
spectrophotometer
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Gel Results
 Compare the samples by eye to the standards
 Spectrophotometers can be used to take absorbance readings at
595 nm
 Graph the controls and use to estimate unknown absorbance
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Summary
 Make sure to:
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Record all steps in your notebook
Be careful not to touch cuvettes where light passes through
Mix samples thoroughly
Compare visually the known and unknown samples that have
been mixed with Bradford reagent
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