Using Spectrophotometer to Measure Protein Concentration Biotechnology

Bellwork List uses of spectrophotometer in Biotechnology.

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

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

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

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

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

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

Detection of Protein with Bradford Reagent

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

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

Example of Standard Curve using Bradford Reagent

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

Determining Unknown Protein concentration

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

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

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

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. 26. 12 Word Summary in 12 words or less, summarize the most important aspects from today's lesson teacher uses a strategy to check all PROPERTY OF PIMA COUNTY JTED, 2010 18