Cell bio module 3

• This process involves rupturing cellular biomembranes to get these proteins out.
• Reasons why a researcher may want to extract and purify their protein of interest from cells

  • Study the folded three‐dimensional structure of the protein using X‐ray crystallography
  • Identify the protein's function
  • Determine what are various interacting substrates, and what does substrate binding do?
  • Purify proteins so that you can identify a protein's amino acid sequence, and with that, predict the sequence of the gene that codes for the protein
  • Purify the protein so you can develop an antibody specific to your protein
• There can be tens of thousands of distinct proteins in a cell, so the main challenge is to separate your protein of interest from all of the other proteins in the entire cell.
• Steps to isolating your protein of interest

  1. Identify a unique assay or experiment for your protein

  2. Choose a source

  3. Extract the proteins from the cell

  4. Solubilize and stabilize your protein

  5. Fractionate or separate your protein from all of the other proteins

  6. Assess or evaluate the purity of the protein that you have isolated
• Protein assay


  A way of detecting your protein
• Examples of protein assays

  • Measure the enzymatic activity of the protein by looking for the release of product or the use of a substrate
  • Use an antibody that is unique to your protein to monitor for the presence and concentration of your protein
  • Monitor for the presence of your protein through its biological activity, such as binding to a unique substrate like RNA or actin
• Characteristics of a good protein assay

  • It must be unique to the protein you are studying, so that you don't mistake another protein for your protein of interest
• Protein source


  The cells from which you will collect your protein of interest
• Characteristics of a good protein source

  • The protein should be easily obtained and present in large amounts
  • The cell type should be low in proteins that might co‐purify with your protein of interest
  • The cell source should be low in proteases that could destroy your protein of interest
  • Expressing your protein in an alternate cell type, such as expressing a mouse protein in bacterial cells, can ensure you are extracting the greatest amount of your protein of interest
• Methods to break open or lyse cells to release proteins

  1. Chemical lysis

  2. Physical grinding

  3. Ultrasonic sonication
• Protein solubilization


  The process of making a protein soluble in an aqueous extract
• Factors affecting protein solubility

  • pH of the solution
  • Salt concentration
  • Presence of detergents
• Membrane-associated proteins are very difficult to isolate because they are amphipathic (possess both hydrophobic and hydrophilic properties) and not soluble in aqueous extracts.
• Protein stabilization


  The effort to maintain the native structure and prevent degradation of a protein during the extraction process
• Factors to consider for protein stabilization

  • Maintaining non-covalent interactions that stabilize the folded conformation
  • pH of the solution
  • Salt concentration
  • Presence of co-factors
  • Temperature
  • Adding protease inhibitors
  • Protein concentration
• Fractionation


  The process of separating proteins into different groups or fractions based on their chemical or physical properties
• Properties used to fractionate proteins

  • Charge
  • Size
  • Polarity
  • Solubility
  • Shape
• Fractionation techniques


  • Ion exchange chromatography
  • Gel electrophoresis
  • Gel filtration chromatography
  • Ultracentrifugation
  • Adsorption chromatography
  • Hydrophobic interaction chromatography
  • Affinity chromatography
• A single fractionation technique is generally not sufficient for protein isolation, and a series of fractionation experiments is needed to isolate the protein of interest.
• Differential centrifugation


  1. Spin the extract at 1000g to pellet nuclei and chloroplasts

  2. Spin the supernatant at 10,000g to pellet mitochondria

  3. Spin the supernatant at 100,000g to pellet membranous organelles

  4. The final supernatant contains soluble, cytosolic proteins
• Chromatography


  A technique where an aqueous extract is poured into a column containing a matrix or beads that help sort proteins based on different properties
• Ion exchange chromatography


  1. Load the protein extract onto a column with charged beads

  2. Positively charged proteins flow through quickly, negatively charged proteins bind to the beads

  3. Elute the bound proteins by disrupting the ionic interactions, e.g. with a salt solution or pH change
• Ras protein


  A small, globular, monomeric GTP-binding protein with GTPase activity, involved in cell signaling and cell cycle pathways
• Gel filtration/size exclusion chromatography

  1. Load the protein extract onto a column with beads containing small cavities

  2. Small proteins get trapped in the cavities, large proteins flow through

  3. Wash the beads to elute the trapped small proteins
• Gel filtration beads have dimpled surfaces with pores of varying sizes, not neatly ordered.
• Affinity chromatography


  1. Load the protein extract onto a column with beads covalently attached to an antibody

  2. The protein of interest binds to the antibody, other proteins flow through

  3. Elute the bound protein by disrupting the antibody-protein interaction, e.g. with pH, temperature or salt changes
• Beads in size exclusion chromatography

  • Dimples across the surface
  • Pores not neatly ordered and not all the same size
  • Defined based on largest protein that could fit
• Affinity chromatography


  1. Beads covalently attached to antibody

  2. Protein of interest binds to antibody, others flow through

  3. Protein of interest eluted by disrupting non-covalent interactions
• Chromatography generally separates proteins in their folded form
• SDS-PAGE electrophoresis


  1. Proteins denatured and coated with SDS to give negative charge

  2. Proteins separate based on molecular weight as they move through gel matrix
• SDS-PAGE gel


  • Lane 1: All proteins after differential centrifugation
  • Lane 2: Fraction after ion exchange chromatography
  • Lane 3: Fraction after gel filtration chromatography
  • Lane 4 & 5: Single 40 kDa protein after affinity chromatography
• Specific activity


  Total enzyme/protein activity divided by total amount of protein
• Protein purification process


  1. Precipitation

  2. Ion exchange chromatography

  3. Size exclusion chromatography

  4. Affinity chromatography
• Protein purification is a multi-step process
• Light microscopes can visualize most prokaryotic cells and organelles in eukaryotic cells
• Electron microscopes can visualize small bacteria, viruses, large proteins, protein complexes, ribosomes, lipids, small molecules
• Resolution (D)


  Smallest distance between two objects at which they still appear distinct
• Numerical aperture (NA)


  Measure of how light is bent as it passes from microscope objective to specimen
• Smaller wavelength of illumination improves resolution
• Microscopy techniques and resolution

  • Brightfield microscopy: ~0.2 μm resolution, 1000x magnification
  • UV microscopy: ~200-300 nm resolution
  • Electron microscopy: 0.1-10 nm resolution