MCB 253

Protein Quantitation

© Elizabeth A. Good
University of Illinois at Urbana-Champaign
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 What is the concentration of our
protein sample?

 What do you get from protein purification?

 Pure protein sample

 What do you do with your pure protein sample?

 Run experiments

 Why quantify before running experiments?

 What does quantitation tell us about the protein sample?

 How much protein you have/concentration

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What properties of proteins are
utilized for quantitation?

 Different molecules absorb different wavelengths.

 UV Spectrophotometry  Proteins absorb UV

 280nm (Aromatic), 200nm(peptide)

 Bradford Assay
 Coomassie blue dye binds to Arg, Aromatic amino acids → Turns blue
 Dye absorbs at 595nm

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 Spectrophotometry

 The absorbed light is related to concentration.

 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

 Set single wavelength at 595nm

 Optical beam

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 Bradford Assay

 Why dilute the protein samples?

 Importance of Bradford Reagent to protein sample ratio (50:1)

 Total sample volume depends on cuvette size = 1.2ml = 1200ul

 Volume of diluted protein sample (protein+buffer) ~ 24ul

 Volume of Bradford Reagent up to 1.2ml

 Each sample will contain 1.2ml Bradford Reagent plus 24ul protein dilution.

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 Calculations of Dilutions

 Concentration,

 C2 = C1
 ;

 Use any ONE of these! They are all the same.

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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)

 Use varied dilutions – why?

 Use a blank – why is it important?

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 Analyzing the Data-
Creating a Standard Curve
(Absorbance vs. Concentration)

 From the spectrophotometer, what

does each concentration give us?

Absorbance
 Absorbance
Line of
 Plot Absorbance vs. Concentration best fit
(ug/mL) for BSA standard protein
Standard curve

 Find absorbance of protein samples

(unknown concentration)

 Use standard curve to find out

concentration of your protein Concentration

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 Good Lab Practices

 Thawing reagents – why?

 Mixing the reagents well – why?

 Avoiding Bubbles – why?

 Accurate Pipetting – why?

 Temperatures and times – importance?

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 TA Help Sessions

 Day: Friday

 Time: 9-10AM, 11AM-1PM, 2-5PM

 Location: Online in Zoom

 Information posted on our course Moodle page

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 Upcoming Week 3 Activities

Week 3-Lab: February 5-9, 2024

 Safety protocols must be followed- long pants, close-
toed shoes, and shirts that cover shoulders/midriff.
 Bradford Assay Experiment

 Data Analysis Group Work

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 Upcoming Assignment Deadlines

Due on Friday, February 2, 2024:

 Protein Purification Protocol

 Five Protein Purification Peer-reviewed Research Papers

 Protein Purification References Page

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