1. Using Spectrophotometer to Measure Protein Concentration
Biotechnology

2. Bellwork
List uses of spectrophotometer in Biotechnology.

3. Objectives
List different indicator solutions used for detecting protein in a mixture
Explain how Bradford reagent is used to determine unknown concentration of protein

4. Detection of protein in solution
In visible light, proteins are colorless
Proteins must be mixed with an indicator solution to make the proteins visible to a spectrophotometer

5. Review of Protein Structure
Proteins are composed of amino acids
Amino acids are linked together by peptide bonds
Amino acid structure- everything but R group constant
R groups varies between amino acids
R group determines how the indicator solutions reacts with the protein

6. Indicator Solutions for Detecting Protein
Biuret
Produces violet-purplish color when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions.
Not very sensitive when protein concentrations are low
Lowry method
Combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins.
Produces a blue color
More sensitive to low protein concentration than Biuret method

7. Indicator Solutions for Detecting Protein
Bradford
Dye-binding assay
Involves adding Coomassie Brilliant Blue G250 to protein and monitoring the color change when the dye binds to the protein
Dye binds to primarily basic and aromatic amino acid residues, particularly arginine
Color changes from brown to blue when it binds to protein

8. Indicator solutions can determine protein concentration
Degree of color change is dependent on the number of protein molecules per volume of solution
Color change can be detected with spectrophotometer
Example: Bradford Reagent
Darker the blue color, the higher the protein concentration

9. Detection of Protein with Bradford Reagent

10. PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share
How is Bradford Reagent used to detect proteins in a solution? How is it different from Biuret reagent?
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
PROPERTY OF PIMA COUNTY JTED, 2010 3

11. Creating a Standard Curve of Known Protein Concentrations
Determine the absorbance of known concentrations of protein mixed with Bradford reagent
The absorbance and protein concentration are plotted on a graph
The line created on this graph is referred to as a standard curve

12. Example of Standard Curve using Bradford Reagent

13. Determination of Protein Concentration of Unknown Sample
Compare the absorbance of unknown protein sample with the absorbance of the known protein concentrations on the standard curve
Extrapolate from the standard curve, the protein concentration (number of protein molecules per given volume of solution) of the unknown solution

14. Determining Unknown Protein concentration

15. PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share
Briefly explain how the protein concentration of unknown solution is determined with Bradford reagent.
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
PROPERTY OF PIMA COUNTY JTED, 2010 3

16. Estimating Protein Concentration using UV light
Proteins absorb ultraviolet light lmax of 280 nm
Aromatic amino acids-phenylalanine, tryptophan, tyrosine are the primary reason for this absorbance peak
Method fast
Does not require indicator solution
Does not destroy the protein sample
Absorption versus concentration linear
However, must be careful of other contaminating molecules that absorb UV light interfering with the assay

17. PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share
List some advantages/disadvantages of A280 for measuring protein concentration.
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
PROPERTY OF PIMA COUNTY JTED, 2010 3

18. PROPERTY OF PIMA COUNTY JTED, 2010
20Word Summary 48 37 18 21 14 13 23
In 20 words or less, summarize the most important aspects from today's lesson.
Word Summary
in 12 words or less, summarize the most important aspects from today's lesson
teacher uses a strategy to check all
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