CBL 3

Study Notes

Biuret Method: Measures protein concentration based on cupric ions (Cu²⁺) binding to peptide bonds under alkaline conditions.
Color changes from blue to purple, signifying protein presence; peak absorbance at 540 nm.
Advantages: Simple method, minimal interference from other compounds.
Disadvantages: Less sensitivity in measuring total protein concentration compared to other methods.
Biorad Method (Bradford Assay): Detects proteins through the interaction of Coomassie Brilliant Blue G dye with peptide bonds in phosphoric acid.
Color shifts from brown to blue indicating protein presence; peak absorbance at 595 nm.
Advantages: Sensitive, accurate measurement of total protein concentration, less prone to interference from other substances.
Disadvantages: Variation in dye-protein binding capacity can affect results.

Biuret Protein Assay:
- Set spectrophotometer to 540 nm.
- Generate standard curve for Biuret protein assay.
- Read absorbance from 500x, 100x, and 10x dilutions of sample A.
- Calculate concentrations for unknown diluted samples.
Bradford Protein Assay:
- Set spectrophotometer to 595 nm.
- Generate standard curve for Bradford protein assay.
- Read absorbance of unknown BSA sample.
- Calculate concentration for the unknown BSA sample.

Introduction: Brief overview of measuring protein concentrations.
Materials: List of chemicals and instruments used with their concentrations.
Methods: Concise steps explaining the experimental procedure.
Results:
- Include raw data tables.
- Present standard curve graphs with properly labeled axes and concentration units for both assays.
- Provide equations and calculations for sample concentrations in Biuret and Bradford assays.
Discussion/Conclusion: Analyze results, address unexpected findings, suggest improvements, and discuss the significance of the lab work.
Submission Format: Use the filename format - BIOL3120_Section_Lastname_Firstname_.

Biuret Method: Measures protein presence via the interaction of cupric ions (Cu²⁺) with peptide bonds in alkaline conditions, causing a color change from blue to purple.
Absorbance: Peak wavelength for detection is at 540 nm.
Pros: Simple methodology with low interference from other compounds.
Cons: Less sensitivity compared to the Biorad method; cannot measure total protein concentrations accurately.
Biorad Method: Uses Coomassie Brilliant Blue G dye, which interacts with peptide bonds in phosphoric acid, resulting in a color shift from brown to blue.
Absorbance: Peak wavelength for detection is at 595 nm.
Pros: Easy to perform, highly sensitive, and exhibits low interference from other substances.
Cons: Variability in dye-protein binding capacity can affect results.

Biuret Protein Assay:
- Set spectrophotometer to 540 nm.
- Create a standard curve.
- Measure absorbance for dilutions (500x, 100x, 10x) of sample A.
- Calculate protein concentration for diluted samples.
Bradford Protein Assay:
- Set spectrophotometer to 595 nm.
- Create a standard curve.
- Measure absorbance for unknown BSA sample.
- Calculate concentration of the unknown BSA sample.

Introduction: Overview of protein concentration measurement methods.
Materials: List of chemicals and instruments with concentrations used.
Methods: Brief outline of experimental steps.
Results:
- Raw data organized in tables.
- Standard curve graphs for Biuret and Bradford assays, including correctly labeled axes and concentration units.
- Equations and calculations for sample A concentrations (500x, 100x, 10x) in Biuret assay.
- Equations and calculations for unknown concentration in Bradford assay.
Discussion/Conclusion: Analyze results, discuss unexpected findings, propose improvements, and highlight the significance of the lab.
File Naming: Format for submission is BIOL3120_Section_Lastname_Firstname.