MCB 253 Protein Quantitation
MCB 253 Protein Quantitation
MCB 253 Protein Quantitation
Protein Quantitation
© Elizabeth A. Good
University of Illinois at Urbana-Champaign
S
What is the concentration of our
protein sample?
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What properties of proteins are
utilized for quantitation?
Bradford Assay
Coomassie blue dye binds to Arg, Aromatic amino acids → Turns blue
Dye absorbs at 595nm
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Spectrophotometry
Beer’s Law
(Absorbance = Molar Absorptivity X Concentration X path length)
Path length = 1cm ;
A C
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Spectrophotometer
7 samples in cuvettes
1 blank + 6 protein samples
Standard protein (BSA)
Your protein sample ([unknown])
Optical window on cuvette
Optical beam
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Bradford Assay
Each sample will contain 1.2ml Bradford Reagent plus 24ul protein dilution.
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Calculations of Dilutions
Concentration,
C2 = C1
;
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Experimental Design
Prepare dilutions:
6 dilutions for standard protein (Bovine Serum Albumin protein
(BSA)- known concentration of 2mg/ml)
6 dilutions for your protein (unknown concentration- what we are
trying to determine)
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Analyzing the Data-
Creating a Standard Curve
(Absorbance vs. Concentration)
Absorbance
Absorbance
Line of
Plot Absorbance vs. Concentration best fit
(ug/mL) for BSA standard protein
Standard curve
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Good Lab Practices
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TA Help Sessions
Day: Friday
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Upcoming Week 3 Activities
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Upcoming Assignment Deadlines
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