Protein Purification and Quantitative Analysis Laboratory Notes

LAB 2 – PROTEIN PURIFICATION AND QUANTITATIVE ANALYSIS

  • Introduction to techniques in cell biology and biochemistry for protein purification and characterization.
  • Methods involved:
    • Ion-exchange chromatography for protein separation.
    • Bradford assay for protein quantification.
    • Polyacrylamide gel electrophoresis for evaluating separation efficacy.

Ion-Exchange Chromatography

  • Definition: A type of chromatography that involves separating proteins based on their net charge at a specific pH.
  • Components:
    • Stationary Phase: Inert beads packed in a glass column, which are charged.
    • Mobile Phase: Solution of proteins being separated.
  • Mechanism of separation:
    • Proteins with charges opposite to the stationary phase bind to the resin.
    • Proteins with similar charges pass through (flow-through).
    • Bound proteins elute by modifying buffer composition.
    • Buffers containing salt (e.g., NaCl) compete with proteins for binding sites.
    • Ion-Exchange types:
      • Anion-Exchange: Positive resin binds negative proteins.
      • Cation-Exchange: Negative resin binds positive proteins.

Bradford Assay

  • Purpose: Colorimetric assay to quantify total protein concentration.
  • Principle:
    • Uses the dye Coomassie brilliant blue G-250, which changes absorbance properties when bound to proteins.
    • Absorbance peaks at:
      • 465 nm (unbound dye)
      • 595 nm (binding to proteins)
  • Assay Procedure:
    • Measure absorption at 595 nm with a spectrophotometer.
    • Generate a standard curve using known concentrations of gamma-globulin.
    • Establish correspondence between absorbance and protein concentration.

Experimental Protocol

Part I: Ion-exchange Chromatography
  • Two types of columns:
    • Cation-exchange with BioRex 70 resin.
    • Anion-exchange with BioRex 5 resin.
  • Protein mixtures:
    • β-amylase (pI = 5.2): Digestive enzyme that breaks down starch.
    • Lysozyme (pI = 9.4): Enzyme that breaks down bacterial cell walls.
  • Steps:
    1. Label and prepare tubes for collection (20 total).
    2. Load protein mixture onto the column after equilibrating with glycine buffer.
    3. Collect fractions of protein using glycine buffer followed by elution buffer to recover bound proteins.
    4. Clean up by regenerating the column with glycine buffer after experiments.
Part II: Bradford Assay
  • Prepare protein standards:
    • Use dilutions from a gamma-globulin stock solution.
    • Use formula C1V1 = C2V2 to calculate required volumes for each dilution.
  • Prepare and label microcentrifuge tubes for standards and samples (both column fractions and the protein mixture).
  • Perform Bradford assay:
    1. Add Bradford reagent to each sample and standards.
    2. Measure absorbance at 595 nm for each standard and sample.
    3. Generate a standard curve to determine unknown protein concentrations.

Results and Data Analysis

  • Record and analyze absorbance values to calculate protein concentrations in each fraction.
  • Use y = mx equation with standard curve to determine protein concentration in samples based on A595 readings.