Protein purification part 2

Overview of Experiments

  • Discussion about the experiments conducted last week.

  • Importance of communication among group members regarding the lab work.

  • Reference to a calculation or self-file document posted on Canvas for consulting while calculating activities and protein concentrations in samples.

  • Emphasis on non-prescriptive nature of the document allowing for customization based on specific experiments.

Preparation for Current Week

  • Introduction to the upcoming week's activities expected to be busy.

  • Previous week’s activities with four students and distribution of tasks within groups noted as important for progress.

Review of Week 1 Experiments

  • Importance of communication and collaboration between partners to understand what has been done.

  • The goal was to set up and successfully conduct experiments involving Zepakryl S200 beads for protein separation.

  • Discussion about the structure of columns and characteristics of samples in terms of molecular weight.

Sample Characteristics

  • Plunger and Column Setup:

    • The plunger used to supply the column should rest at the bead level.

    • Loading different analytes (e.g., progesterone and myoglobin) depending on their molecular weights into the column.

  • Molecular Weights:

    • Glutextrin: 2,000 kDa

    • Myoglobin: 17 kDa

    • Estimates for expected elution times based on molecular weights.

Fraction Collection in Columns

  • Importance of observing the density and consistency of fractions collected with Buffer A.

  • Proteins are typically heavier which affects drop size and collection accuracy.

  • Best practice to visually check the collected fractions instead of relying solely on the fraction collector machine.

Technical Considerations

  • Ability to stop the pump when approaching desired fractioning volume is crucial.

  • Required steps in handling fractions post-collection (labeling, capping, and storing properly).

Yeast Preparation Review

  • Steps followed in pelleting samples using 55% ammonium sulfate to saturate samples, noting that ADH is insoluble in high salt concentrations.

  • Each group required to prepare, process, and resuspend the pellet samples for further electrophoresis.

Collection of Samples

  • Specific samples to be generated during the experiments must be documented which include:

    • Sample 1: Total cell homogenate (homogeneous).

    • Sample 2: Cytoplasmic extract.

    • Sample 3: Supernatant after ammonium sulfate precipitation.

    • Sample 4: Pellet sample enriched in ADH.

Protein Profiling and Activity Measurement

  • Running SDS-PAGE gels to observe protein content and purification status.

  • Explanation of typical bands expected from different samples with respect to ADH activity.

Purification Goals

  • The pathway to increasing enzyme purity and enzyme activity as desired outcomes of the experimental protocol.

  • Factors influencing ADH's apparent activity may vary significantly across sample treatment conditions.

Purification Metrics

  • Definitions of important purification metrics discussed:

    • Total activity, specific activity, yield as fractions undergo purification.

    • Ideal outcomes of purification in a visual, tabulated form detailing protein recovery metrics.

Key Definitions

  • Enzyme Activity: Defined as the amount of enzyme catalyzing a transformation of one micromole of substrate to product per minute under specific conditions.

  • Activity measurements often associate with NADH and NAD+ due to their spectrophotometric properties.

Elucidation of Activity Assays

  • Importance of establishing conditions for consistent and repeatable measurements of enzymatic activity in samples.

  • Emphasis on enzyme purity during the purification process aimed at obtaining a functional enzyme for research purposes.

Calculation of Specific Activity

  • Describes how to calculate specific activity based on enzyme assays.

  • Key points discussed:

    • Conducting enzymatic assays requires measuring the reactants and products' concentration changes over time.

    • Emphasis on how absorbance correlates with reaction rates.

Example Calculation Scenarios

  • Working through examples with various dilution factors and concentrations to derive specific activity measurements, strongly advising comprehensive understanding of units involved in activity calculations.

  • Importance of methodical handling and documentation throughout laboratory processes for accurate data collection.

Lab Logistics and Management

  • Outline of expectations for group preparation, including:

    • Making fresh buffers before experiments.

    • Coordinating assays between group members to optimize work efficiency.

    • Importance of labeling samples accurately during experiments to avoid mix-ups.

Closing Remarks

  • Review of technical considerations with regards to using equipment, sample handling, and the necessity of collaborative efforts among team members to successfully navigate the week’s protocols and experiments.