MCB 102 Lec 5 Protein purification

  1. What's the first thing you have to do when you wanna study proteins?
    Gotta obtain it
  2. All proteins come from some source, either _______ or _______.
    • Biological, natural source
    • Bacteria
  3. What do we need to do to understand how proteins function and are structured?
    • Isolate them
    • Sequence them
    • Look at their structure
    • Investigate their chemical properties
    • See how they interact with each other
  4. About how much dry weight of a mammalian cell is taken up by proteins?
  5. What's a proteome?
    The constellation of proteins in a cell
  6. Understanding the function of a protein requires its _______.
  7. What is purification?
    When a complex mixture is resolved or disconvoluted into its constituents by the successive application of physiochemical methods for separation and fractionation
  8. What three things are done after obtaining a single pure protein?
    • Analysis of its AA sequence
    • Examinaton of its functional characteristics
    • Determination of its 3D structure
  9. What physical and chemical properties are observed during methods of protein purification in order to exploit the difference between proteins?
    • Mass
    • Shape
    • Polarity/solubility
    • Charge
    • Density
    • Hydrophobicity
    • Ligand-binding specificity and affinity
    • Special features
  10. What is specific activity?
    The ratio of the amount of the desired protein present to the amount of total protein present in the same sample
  11. How is the amount of desired protein present measured?
    Via its activity in our specific assay
  12. What three things do we need to find the specific activity of a protein?
    • Assays to detect the presence of the protein of interest at each purification step
    • Assays to measure TOTAL protein at each purification step
    • Methods to purify the protein
  13. What are the different biochemical assays to detect the presence of the protein of interest?
    • Colorimetric assays
    • Radiochemical assays
    • Spectrophometric assay
    • Immunochemical assay
    • Biological activity assay
  14. What are the different assays to measure TOTAL protein?
    • Lowry method
    • Bradford method
  15. What is the concept of a biochemical assay?
    • A qualitative or quantitative method for detecting the presence and measuring the amt of a biological substance in a complex mixture of other biomolecules
    • The action of an enzyme leaves a "molecular footprint"
  16. The action of an enzyme leaves a "____________".
    Molecular footprint
  17. What are the two cases of a colorimetric assay?
    • 1. Either the substrate or product (or both) of the reaction have a specific light absorbance that changes as the reaction progresses
    • 2. There is a way to stain for substrate or product after the reaction
  18. What are the two cases of a radiochemical assay?
    • 1. The product of the enzymatic reaction is radioactively labeled
    • 2. A by-product of the enzymatic reaction is radioactively labeled
  19. What is spectrophotometric assay?
    • The absorbance of a product or substrate changes as the reaction progresses
    • The whole reaction takes place in a spectrophotometer
  20. What does immunochemical assay use?
  21. Give an example of colorimetric assay (case 1)
    • Trying to detect the activity of the enzyme β-galactosidase (cleaving enzyme)
    • Galactose is attached to a substance to make the colorless substrate galactoside (X-gal)
    • When the substrate is cleaved, it turns blue
    • Shows that β-galactosidase was there to cleave
    • Concentration of product is assayed by absorbance at 570 nm
    • Want excess substrate so amt's of product and concentration of enzyme are linear
    • Map enzyme concentration vs. absorbance on a standard curve
  22. What's a standard curve used for?
    Used as a reference
  23. Give an example of colorimetric assay (case 2)
    • PI-Scel endonuclease (cuts DNA?)
    • Stain DNA with dye that absorbs UV light and emits visible red light
  24. Give an example of radiochemical assay for an enzyme (case 1)
    • Hexokinase (glucose phosphorylation; transfers phosphorous group from ATP to RADIOACTIVE glucose)
    • Now glucose has a NEGATIVE charge
    • DEAE positively charged cellulose filter paper
    • ATP, ADP, and P-glucose will bind to filter; glucose will pass
    • Find radioactive group stuck to filter using scintillation counter
    • Quantify P-glucose
    • Compare and map to standard curve
  25. Give an example of radiochemical assay for an enzyme (case 2)
    • Theymidylate synthase (enzyme)
    • Creates water through condensation
    • Receives radioactive/heavy proton from substrate
    • Filter through activated charcoal
    • All organic molecules will bind, but radioactively labeled water will pass
    • Measure activity of enzyme by assessing amt of radioactive water that filtered through charcoal
  26. Give an example of spectrophotometric assay for an enzyme (case 1)
    • Lactate dehydrogenase (reduces NAD+ and produces NADH)
    • Look for changes in absorbance from light passing through substances
    • Because enzyme uses NADH as a substrate, its concentration will decrease according to enzyme concentration
  27. Describe immunochemical assay for protein detection (case 1)
    • HIV coat proteins in human serum
    • a. Standard enzyme-linked immunosorbant assay (ELISA)
    • Antigen → Add specific antibody → Add enzyme-linked antibody → add substrate, get yellow
    • Only get reaction if enzyme present; Enzyme only present if antibody binds
    • b. Sandwich ELISA
    • First antibodies → Add antigen → Add second enzyme-linked antibody → Add substrate, get yellow
  28. Give an example of immunochemical assay for protein detection
    • p-nitrophenyl-phosphate (colorless substrate)
    • Alkaline phosphatase (antibody coupled enzyme)
    • p-nitrophenol (bright yellow product)
    • Can also be seen as colorimetric
  29. What are the different biochemical ways to assay TOTAL protein in a sample?
    • Lowry Method
    • Bradford method
  30. What is Lowry method?
    • Named for the biochemist Oliver H Lowry
    • His 1951 paper describing the technique is most-highly cited
    • Better if working with membrane potentials
    • Not good in cytosol b/c sensitive to the reducing agents normally found there
  31. What is Bradford method?
    • Developed by Marion M Bradford (1976)
    • Requires very low concentrations of protein (thus samples usually have to be diluted)
    • Sensitive to detergents
    • Better for cytosolic proteins
  32. What is the biuret test?
    • Chemical test for detecting the presence of peptide bonds
    • In the presence of peptides, a copper2+ ion forms violet-colored coordination complexes in an alkaline soln
  33. Why can the biuret reaction be used to assess the concentration of proteins?
    B/c peptide bonds occur with the same frequency per amino acid in the peptide
  34. The intensity of color (absorption at 540nm) in a biuret test is directly proportional to what?
    Protein concentration
  35. The intensity of color in a biuret test is ________ proportional to the protein concentration.
  36. What is directly proportional to the concentration of proteins in a biuret test?
    The intensity of color
  37. The Lowry method for protein determination combines what two tests?
    • The Biuret test (the rxn of Cu2+ ions with the peptides under alkaline conditions)
    • The oxidation of aromatic protein residues using the Folin-Ciocalteu reagent
  38. How does the oxidation of aromatic protein residues using the Folin reagent affect sensitivity?
    Increases it 100 times
  39. The biuret test is unique to which method for protein determination?
    Lowry method
  40. What kind of proteins are used in the Bradford method?
    Proteins with basic and aromatic side chains
  41. For most methods, what do you need to make to compare your unknown sample to?
    A standard curve
  42. Every protein purification takes place in what?
    A buffer
  43. What is a protein purification buffer?
    • Aqueous mixture of components that:
    • -- extract the desired protein into solution
    • -- helps preserve the structure and function of the desired protein
  44. How does a protein purification buffer help preserve the structure and function of the desired protein?
    • It resists any change in pH
    • It contains other reagents that help prevent the desired protein from suffering unwanted oxidative and/or proteolytic damage
  45. How do protein purification buffers resist any change in pH?
    It contains equimolar amounts of a conjugate acid-base pair that has a pKa value at or very near phisiological pH
  46. What are some typical buffer components used in protein purification?
    • Buffering species
    • Salt
    • Reducing agent
    • Chelator
    • Protease inhibitor
    • Other
  47. Why are buffering species used in buffers for protein purification?
    To hold pH constant
  48. Why is salt used in buffers for protein purification?
    • Proteins are more soluble at moderate ionic strength
    • Helps maintain dielectric constant
  49. Why are reducing agents used in buffers for protein purification?
    To prevent oxidation of Cysteine
  50. Why are chelators used in buffers for protein purification?
    To remove heavy metal ions
  51. What are chelators?
    Substance bonded to a metal ion at two or more points
  52. Why are protease inhibitors used in buffers for protein purification?
    To revent proteolysis
  53. As purification of proteins proceeds, what is being removed?
    Extraneous proteins and other contaminants
  54. What rises as the purification of protein proceeds?
    Specific activity
  55. When does specific activity eventually reach a maximum?
    As purification of protein proceeds till the end
  56. What is one measure of protein purity?
    When specific activity remains constant and cannot be further increased
  57. What do you use to isolate proteins from specific subcellular fractions/organelles?
    Differential centrifugation
  58. What is differential centrifugation used for?
    To isolate proteins from specific subcellular fractions/organelles
  59. What is differential centrifugation?
    • Crush/blend tissue completely
    • Centrifuge at low speed and separate supernatant
    • Repeat but centrifuge at higher speed than before
    • Repeat until supernatant is relatively clear
    • Supernatant contains soluble proteins
    • What steps you take depends on your protein
  60. What are the steps in differential centrifugation?
    • Break open the cell
    • If the protein is in the cytosol, the crude lysate may be filtered or centrifuged to remove the cell debris and stuff; Protein of interest is in the supernatant
    • If the protein is in a membrane or organelle, repeated centrifugation and isolation of pellet or supernatant will provide access to the organelle or membrane
  61. How would you break open an animal cell for differential centrifugation?
    Place cell in hypotonic/osmotic solution
  62. How would you break open a plant/bacteral cell for differential centrifugation?
    • They have cell walls
    • Blender
    • Sonicator (ultrasound)
    • French press
  63. What steps do you have to take for differential centrifugation if the protein is in the cytosol?
    • After cells have been broken, the crude lysate may be filtered or centrifuged to remove the cell debris
    • The protein of interest is in the supernatant
  64. What steps do you have to take for differential centrifugation if the protein is in a membrane or organelle?
    Repeated centrifugation and isolation of the pellet or supernatant will provide access to the organelle or membrane
  65. How do we get the protein from the supernatant?
    • We need to know something about the protein
    • Some proteins will precipitate if we add salt (usually ammonium sulfate) → "Salting out"
    • If we know the approximate size of the protein, we can use a dialysis process with a membrane of the right size
    • Column chromatography can separate proteins according to charge, size, affinity for a specific ligand, etc.
  66. What is salting out?
    • After proteins solubilized, they can be purified based on solubility
    • Use ammonium sulfate (NH4SO4) as salt
    • Takes away water by interacting with it
    • Makes protein less soluble b/c hydrophobic interactions among proteins increases
    • Aggregation of proteins by their most hydrophobic surfaces b/c no tenough free H2O for solvation
    • Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments)
    • One fraction has the protein of interest
  67. Regardless of the specific type of chromatography, What should you always do to get a protein from supernatant?
    • Start with a sample of our protein in soluble form
    • Run it through a column
    • Collect fractons at different times
    • Use a biochemical assay to identify the fraction containing our protein
  68. What are the two phases of columns in column chromatography?
    • Stationary
    • Mobile
  69. What is a stationary phase in column chromatography?
    • Solid porous matrix
    • Protein samples go through and interact with this phase
  70. What is a mobile phase in column chromatography?
    • Protein sample
    • Flows over the stationary phase
    • Carries along with it the sample to be separated
  71. What are the steps of column chromatography?
    • Start with a sample of our protein in soluble form
    • Use a buffer/soln to "push" our protein (mobile phase running down column)
    • Proteins will be separated as they will run at different speeds depending on their properties
    • Collect fractions of eluate at different times
  72. What is effluent?
    What you collect during column chromatography
  73. What is size exclusion chromatography?
    • Gel filtration
    • Proteins are separated by size
  74. What is the process for size-exclusion chromatography/gel filtration?
    • Column contains a cross-linked polymer
    • Polymer contains small pores
    • Proteins that are small enough to enter the pores will be stuck and delayed
    • Larger molecules pass more freely; apprearing on the earlier fractions
  75. What is ion-exchange chromatography?
    Separates protein according to charge
  76. What is the process for ion-exchange chromatography?
    • Column contains cation or anion exchangers (polymers w/ charged functional groups)
    • Proteins move through the column at rates determined by their net charge at the pH used (and elution buffer used)
    • With cation exchangers, proteins with a more negative net charge move faster and elute earlier as they are repelled
  77. What is affinity chromatography?
    Based on ligand binding affinity
  78. What are the first steps of affinity chromatography?
    • Beads have a ligand covalently attached that will bind the protein of interest and retard its migration through the matrix
    • Unwanted species will wash through the column
  79. What are the second steps of affinity chromatography?
    • After unwanted proteins have washed
    • A solution containing the same ligand is now added to the column (could also be high salt or different pH to disrupt interactions)
    • The protein is eluted by the ligand solution
  80. What does eluted mean?
    Remove by washing withsolvent
  81. What is hydrophobic interaction chromatography (HIC)?
    • Relatively new technique
    • Takes advantage of the fact that most proteins have hydrophobic pockets and of the hydrophobic effect
    • Matrix of the column consists of hydrophobic ligands
    • Protein solution is mixed with high levels of salt, promoting hydrophobic effect (but not too high b/c can cause ppt)
    • Proteins will elute according to their hydrophobicity, with the most hydrophobic proteins beind delayed the most
    • Elution is done with solutions of DECREASING salt concentration
  82. What happens if the salt levels are too high in the protein solution during HIC?
  83. How are chromatographic methods typically enhanced?
    Using high performance liquid chromatography
  84. What is high performance liquid chromatography?
    • HPLC
    • Uses high pressure pumps that speed up mvmt of the protein molc down the column
    • Speical columns that can sustain the pressure of the pressurized flow (no compressible resin and strong columns)
    • Results in reduced transit time which limits the diffusion of the protein and enhances the resolution
  85. What does HPLC result in?
    • Reduced transit time
    • Limits the diffusion of protein
    • Enhances resolution
  86. When is electrophoresis used?
    For protein analysis
  87. How is separation in analytical scale commonly achieved?
    By electrophoresis
  88. What is electrophoresis?
    • Electric field pulling on proteins according to their charge
    • Gel matrix hinders mobility of proteins according to their size and shape
  89. Why is electrophoresis useful?
    • Allows us to visually separate proteins and assess the degree of purity of a mixture
    • Can also determine the MW and pI of proteins
  90. What is polyacrylamide gel electrophoresis?
    • Different samples are loaded in wells or depressions at the top of the polyacrylamide gel
    • The proteins move into the gel when an electric field is applied
    • The gel minimizes protein mvmts other than those induced by the electric field
  91. The migration of proteins in a gel is a function of what?
    Their size and shape
  92. How can proteins be run in gels?
    • In their native form
    • Bound to sodium dodecyl sulfate (SDS)
  93. How does SDS allow proteins to run in gels?
    • Denatures the protein (no 3D structure interference)
    • Makes the protein negatively charged with overall charge being proportional to the length of the protein
    • Rate of mvmt will only depend on size, with smaller polypeptides migrating more rapidly
  94. When SDS binds to proteins, why is the overall charge proportional to the length of the protein?
    Proteins bind approximately 1 molecule of SDS/AA
  95. What must be mixed with proteins to run a gel?
    Gel Loading Buffer
  96. Why must a gel loading buffer be mixed with proteins to run a gel?
    • It has glycerol (density)
    • It has bromophenol blue (dye)
  97. Why is glycerol in gel loading buffer useful to run a gel?
    • Density
    • Need the protein to stay at bottom of well/groove until it's time for electricity
  98. Why is bromophenol blue in gel loading buffer useful to run a gel?
    • It's a dye that can be seen by the naked eye
    • Used to monitor the migration of proteins in the gel
  99. What is a ladder?
    Has proteins of different known sizes
  100. Why are ladders used when running a gel?
    Used to estimate the size of the protein
  101. What are the final steps of SDS-PAGE?
    • Once the gel has run, it's placed in a Comassie Blue soln that binds to proteins
    • Each band in the gel represents a different protein or protein subunit
    • Can see different purification steps
  102. SDS-PAGE can be used to estimate what about the protein?
    The molecular weight
  103. What is isoelectric focusing?
    A procedure used to determine the isoelectric point of a protein
  104. How is the pI of a protein determined using isoelectric focusing?
    • A pH gradient is established in a gel
    • When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI
  105. What is two-dimensional electrophoresis?
    • Isoelectric focusing and SDS-PAGE are combined
    • Proteins are first separated by isoelectric focusing in a thin strip gel
    • The gel is tehn laid horizonally on a second, slab-shaped gel (SDS-PAGE)
    • The proteins are separated by SDS polyacrylamide gel electrophoresis
    • Thus, the original protein mixture is spread in two dimensions
  106. What does horizontal separation show in 2D electrophoresis?
    Reflects differences in pI
  107. What does vertical separation show in 2D electrophoresis?
    Reflects differences in molecular weight
  108. What important things can be compared using 2D gels?
    • Protein pools
    • (treatment, diseases, etc.)
Card Set
MCB 102 Lec 5 Protein purification
MCB 102 Lec 5 Protein purification