Cell Lab 3.pptx

BIOL 3120 (Cell Biology Lab) Lab 3 - Measuring Protein Concentration Rubaia Tasmin Teaching Assistant 09/20/2024 Email: [email protected]  Contents 1.Principals of Biuret and Bradford protein assays 2.Today’s Lab Proc...

BIOL 3120 (Cell Biology Lab) Lab 3 - Measuring Protein Concentration Rubaia Tasmin Teaching Assistant 09/20/2024 Email: [email protected]  Contents 1.Principals of Biuret and Bradford protein assays 2.Today’s Lab Procedure 3.Lab 3 Report Requirements 2  Protein assay using Biuret method  Involves the interaction between cupric ions (Cu²⁺) and the peptide bonds in proteins under alkaline conditions  Color changes from blue to purple, indicating the presence of proteins  Peak absorbance wavelength is at 540 nm  Advantages: - Simplest protein assay method - Has low interference with other compounds  Disadvantages: - Not as sensitive as the Biorad method (cannot accurately measure the concentration of total proteins)  Qualitative protein test: Biuret Method Presence Absence of of proteins proteins https://biologynotesonline.com/biuret-test-for-protein-principle-procedure-result-application/#princi ple_of_biuret_test  Protein assay using Biorad method  Involves the interaction between a dye (Coomassie Brilliant Blue G) and the peptide bonds of proteins in phosphoric acid  The dye color changes from brown to blue, indicating the presence of proteins  Peak absorbance wavelength at 595 nm  Advantages: - Easy to perform - Very sensitive (can accurately measure the total protein concentration) - Less subject to interference with other compounds  Disadvantages: - The dye-protein binding capacity varies greatly depending upon Quantitative protein test: Bio- Rad Method Absence Presence of of proteins proteins https://www.sciencedirect.com/science/article/pii/ S0003269718301994  Today’s Lab Procedure Biuret protein Bradford protein assay assay 1. Set the spectrophotometer 1. Set the spectrophotometer wavelength to 540 nm wavelength to 595 nm 2. Generate a standard curve for 2. Generate a standard curve for Biuret protein assay Bradford protein assay 3. Read the absorbance of 500x, 3. Read the absorbance of 100x and 10x dilutions of sample unknown BSA sample A 4. Calculate the concentration of 4. Calculate the concentration of the unknown BSA sample the unknown diluted samples 7  Requirements for Lab 3 Report 1. Introduction: Introduction of protein concentrations measuring. 2. Materials: All the chemicals used along with concentration, and instruments used. 3. Methods: Brief steps for the experiment you did. 4. Results: Raw data in tables. Standard curve graphs including suitable axes name and concentration units for both Biuret and Bradford protein assays. Standard curve equation and calculations for Sample A concentration (500x, 100x, and 10x dilutions) in Biuret assay. Standard curve equation and calculations for the unknown concentration in Bradford assay. 5. Discussion/conclusion: Interpret your results. Discuss unexpected findings. What improvements can you make? What is the significance of the File name for your lab report submission- lab? BIOL3120_Section_Lastname_Firstname_ 6. Lab manual questions & underlined questions LabN​ 8