Week 25 BIOS5320 Pre-practical for Week 26

Mini-Project 2 Overview

Pre-practical preparation for Week 26

Procedures in Week 26 Practical:

1. Lactase Assay

   • Detailed lab procedures for conducting the lactase assay.

   • Emphasis on data analysis methods post-experimentation.

   • Understanding of both Michaelis-Menten plot and Lineweaver-Burke plot to determine kinetic parameters.

Important reminders:

• Maintain meticulous records in the lab book, critical for assessment which will occur during Week 26 practical session.

Feedback from Previous Sessions

• Positive feedback noted on demonstrators and techniques, particularly concerning:

  • Helpfulness and engagement of demonstrators, fostering an interactive learning environment.

• Suggestions for improvement included:

  • More detailed instructions for laboratory procedures.

  • Longer breaks before lunch to enhance focus during afternoon sessions.

• Highlighted challenges faced:

  • Techniques like SDS-PAGE which require precision and understanding of molecular weight assessments.

  • Importance of sharing reagents and equipment among peers to optimize lab efficiency.

Achievements from Week 24 Practical

• Completed experiments include:

  • Protein extraction from supplements A-D, ensuring comprehensive collection of proteins for analysis.

  • Lactase protein concentration determination using the Bradford assay, facilitating accurate quantification of lactase levels.

  • SDS-PAGE gel analysis conducted for determining molecular weight and purity of supplements, crucial for understanding supplement quality.

Week 24 Data Analysis

• In lab:

  • Successfully uploaded protein concentration data (contributing 2% to the overall mark).

  • Class data available on Moodle under Mini Project 2 for comparison and collaborative analysis.

• At home tasks:

  • Creation of Excel-based BSA (Bovine Serum Albumin) standard curve with error bars and linear equation for future reference.

  • Calculation of extract concentrations and averaging class-wide concentration data for comprehensive insights.

  • Annotation of SDS-PAGE gel image to mark significant findings visually.

  • Plotting molecular weights of markers to determine the molecular weights of supplements by comparison.

Key Data to Complete

Completed Data and Analysis for Week 26:

• Determination of lactase extract concentrations for supplements A-D crucial for efficacy assessment.

• Molecular weight determination via SDS-PAGE, supporting findings in protein characterization.

• Annotated SDS-PAGE gel image will serve as a visual summation of molecular weight results.

• Excel-generated standard curve graph for molecular weight markers for future experimental contexts.

• Determination of Vmax and Km values for lactase extracts to establish enzyme kinetics characteristics.

• Individual Michaelis-Menten and Lineweaver-Burk graphs for each supplement to visualize kinetic data effectively.

• Compilation of class averages and pH impact graphs to correlate varying conditions on enzyme performance.

Upcoming Investigation (Week 26)

Investigation Focus:

• Substrate concentration variation to determine the optimal lactase supplement for application.

Important parameters:

• Km: Represents the substrate concentration at which the enzyme operates at half its maximum velocity, providing insights into the binding affinity of the enzyme.

• Vmax: Indicates the maximum catalytic rate achieved when the enzyme is saturated with the substrate, critical for understanding enzyme efficiency.

• Experiment will utilize a lactase assay with an alternate substrate (ONPG) to ascertain broader applicability.

Lactase Assay Details

• While lactose is the primary substrate, ONPG will be employed instead, expanding substrate scope.

• The reaction yields D-galactose and the yellow compound ortho-nitrophenol, which will be measured at 405 nm for quantification of activity.

Lab Preparation Steps

• Secure protein sample from week 24 (initially keep on ice to preserve enzyme activity).

• Set up and calibrate Spectrophotometer for accurate 405 nm readings crucial for determining absorbance and reaction rates.

Health, Safety, and Waste Disposal

• Adhere strictly to health and safety protocols outlined in Moodle to ensure safe laboratory practice.

• Always wear gloves and maintain a clean work area to prevent contamination.

• Ensure all samples are appropriately labeled to avoid any mix-up or errors during analysis.

Conducting the Assay

• Sample preparation must include:

  • Addition of assay buffer and ONPG to a cuvette carefully measured for accuracy.

  • Rapid addition of lactase extract followed by immediate monitoring of absorbance to track the reaction.

• Aim for an initial absorbance change rate of 0.25 OD/min to ensure effective measurement of enzyme activity.

Analyzing Initial Rates

• Continuous monitoring of absorbance changes to calculate accurate reaction rates necessary for enzymatic analysis.

• Ensure constants in absorbance values indicate steady rates to confirm reliability of data.

• Avoid manual data recording; utilize automated systems for increased accuracy and efficiency.

Rate Adjustment Procedures

• If the OD rate is too high, options include dilution of protein or adjusting enzyme volume accordingly.

• If the OD rate is too low, evaluate for potential protein denaturation and adjust conditions as necessary to restore activity.

Substrate Concentration Investigation

• Vary ONPG concentration systematically from 3 mM to 0.1 mM and thoroughly document concentrations utilized for detailed analysis.

• Maintain a consistent assay volume of 1 ml across experiments to ensure comparability of results.

• Conduct assays in triplicate to validate results and ensure reliability of experimental data.

Final Data Submission

• Submit the average reaction rates for varying ONPG concentrations (3, 1.5, 0.75, 0.375, 0.185 mM) before leaving the lab to contribute to group analysis.

• Attendance registration is crucial for record-keeping.

Data Conversion for Reporting

• Convert raw data for conventional reporting:

  • Transition volumes to concentrations (M, mM, µM) for clarity.

  • OD/min values must be converted to µmoles/min for standardized reaction rate reporting.

Beer-Lambert Law for Conversion

• Use the OD change/min value along with the molar extinction coefficient for accurate calculations.

• Important notes on formula application to ensure conversion accuracy.

Data Sorting Example

• Include hypothetical sorting values to demonstrate conversion results from OD to nmoles/min for practical understanding.

Graph Plotting

• Utilize both Michaelis-Menten and Lineweaver-Burke plots for comprehensive Vmax and Km determination.

• Follow specific plotting and calculation instructions detailed in the session to ensure precision in results.

Summary of Coverage

• Recap covered practical achievements from Week 24, laying the groundwork for Week 26.

• Detailed procedures outlined for upcoming practicals to optimize learning outcomes.

• Emphasis on data analysis and report preparation techniques crucial for successful project completion.