BIOCHM E2 C5

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BIOCHM E2 C5
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2011-10-10 10:26:42
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BIOCHM E2 C5
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  1. WHY PURIFY AND ID PROTEINS?
    • STRUCTURE
    • BIOCHM/PHY PROPERTIES
    • BIO FUNCTIONS
  2. DEFINE PROTEOME
    ALL THE PROTEINS IN A CELL
  3. NAME THE STEPS TO STUDY A PROTEIN.
    • 1.ISOLATE
    • 2. SOLUBILIZE
    • 3. PURIFY
    • 4. DETECT
  4. NAME 5 TYPES OF ISOLATION TECS THAT YOU USE TO ISOLATE PROTEIN FROM A CELL TO STUDY IT.
    • 1. CHEM LYSIS. HYDRPHOBIC SOLVANTS TAKE AWAY H20 FROM CELL WALL.
    • 2. ENZYMES. LYSOZYMES CELL AT WALL.
    • 3.MECHANICAL LYSIS..WATER TO POP CELLS. FRENCH PRESS.
    • 4. SONICATION
    • 5. GRINDING.--> MOTOR & PESTTLE.
  5. WHEN ONE SOLUBILIZES A PROTEIN, WHAT IS THE MAIN FACTOR THAT MUST BE DONE.
    • OPTIMIZE BUFFER pH (pI POINT), SALT CONCENTRATION, TEMP, CHARGE.
    • KEEP PROTEIN HAPPY.
  6. NAME 3 PURIFICATION TECS.
    • 1.COLUMN CHROMATOGRAPHY
    • 2. CENTRIFUGATION (SEC, IEC, AFFINITY)
    • 3.PRECIPITATION/ DIALYSIS
  7. NAME 5 METHODS TO DETECT THE PRESENCE OF PROTEINS IN EXTRACT.
    • ELECTROPHORESIS
    • MASS SPECTROMETRY
    • WESTERN BLOT
    • SDS-PAGE
    • SEC
  8. DO THE PROTEIN FLOWCHART
  9. AFTER LYSIS OF CELL, NAME 3 WAYS TO ISOLATE PROTEIN FROM SOLUTION.
    • 1. CHARGE
    • 2. SIZE
    • 3. SPECIFICITY (TO Ab OR SPECIFIC TAG) BEST WAY.
    • 4. LOCATION & ABUNDANCE. IF YOU WANT TO STUDY PROTEIN BRAIN CELLS, GET THEM FROM THE BRAIN, NOT THE LIVER.
  10. DEFINE MOBILE & STATIONARY PHASES IN BASIC CHROMMATOGRAPHY.
    • MOBILE IS THE LIQUID THAT MOVES THRU.
    • STATIONARY IS THE PACKING, RESIN OR BEADS.
    • NOTE THAT COMPOUNDS THAT INTERACT MORE WITH THE STATIONARY PHASE ARE RETAINED LONGER AND "ELUDE" LATER.
  11. T OR F?
    PROTEINS WITH SIMILAR PROPERTIES MAY BE MORE DIFFICULT TO ISOLATE AND PURIFY & OFTER REQUIRE MULTIPLE TECHINQUES.
    TRUE
  12. LABEL THE UNITS
  13. IN ION EXCHCHANGE CXHROMATOGRAPHY, WHAT IS THE PRIMARY OBJECTIVE?
    SEPERATION BY CHARGE.
  14. IN IEC, DEFINE THE ANION EXCHANGE FORMULA.
    WHAT TYPE OF ION WILL IT ATTRACT?
    • R+ A- + B- <---> R+ B- + A-
    • NEGATIVELY CHARGE PROTEINS.
    • THE RESIN CONSIST OF POSIVE CHARGE MATERIALS THAT ATTRACH THE NEGATIVE IONS OF THE PROTEIN.
  15. SEPERATION IN ION EXCHANGE CHROMATOGRAPHY DEPENDS UPON
    • THE REVERSIBLE ABSORPTION OF CHARGED SOLUTE MOLECULES TO
    • IMMOBILIZED ION EXCHANGE GROUPS OF OPPSITE CHARGE.
  16. R+ A- + B- <--> R+B- + A-
    WHAT TYPE OF IEC IS THIS AND DEFINE THE UNITS.
    • ANION EXCHANGE.
    • THIS WILL ATTARCH NEGATIVELY CHARGED PROTEINS.
    • R+= EXCHANGE ON RESIN "STATIONARY PHASE"
    • A- = COUNTER ION (SALT ION Na+ OR Cl-)
    • B - = PROTEIN. IN THIS CASE, IT HAS A NEGATIVE CHARGE.
  17. WHAT'S THE FIRST THING YOU DO WHEN ONE WANTS TO RUN A ION EXCHANGE CHROMATOGRAPHIC TEST?
    CALCULATE THE pI
  18. GIVE AND EXAMPLE OF AN ANION USED IN ION EXCHANGE CHROMATOGRAPHY.
    • DEAE
    • DIETHYLAMINO ETHYL (BASIC GROUP)
  19. GIVE AND EXAMPLE OF AN CATION USED IN ION EXCHANGE CHROMATOGRAPHY.
    • CM
    • CARBOXYMETHYL (ACIDIC GROUP)
    • -CH2COO-
  20. THE NOMACLATURE OF THE ANION/CATION EXCHAGE IN ION EXCHANGE CHROMATOGRAPHY IS BASED ON WHAT?
    IF YOU WANT TO SEPERATE A POSITIVELY CHARGE PROTEIN, YOU WOULD USE A CATION EXCHANGE AND VISA VERSA FOR A NEGATIVE ONE.
  21. R-A+ + B+ <-->R- B + + A+

    WHAT TYPE OF ION EXCHANGE IS THIS AND DEFINE THE UNITS.
    • CATION EXCHANGE.
    • THIS WILL ATTARCH POSITIVELY CHARGED PROTEINS.
    • R-= EXCHANGE ON RESIN "STATIONARY PHASE"
    • A+ = COUNTER ION (SALT ION Na+ OR Cl-)
    • B + = PROTEIN. IN THIS CASE, IT HAS A POSITIVE CHARGE.
  22. IF YOU USED CARBOXYL METHYL IN THE ION EXCHANGE CHROMATOGRAPHY, WHAT COULD YOU SAY ABOUT THE PROTEIN.
    THE PROTEIN IS BASIC, BECAUSE THE CARBOXYL METHYL IS BASIC. ALSO THAT IT WOULD TAKE MORE SALT TO DISPLACE THE PROTEIN BECAUSE IT'S MORE TIGHTLY BOUND.
  23. HOW DOES ONE MONITOR FRACTIONS DURING SEPERATION?
    • LIGHT ABSORBANCY.
    • ABS=CONCENTRATION.
    • PROTEINS USUALLY CONTAIN ONE AROMATIC AA THAT ABS LIGHT.
    • 220= ALOT OF THINGS
    • 280 = TYR & TRP
    • 254 = PHE
  24. WHAT IS THE SIZE OF INSULIN IN kD?
    5.7 kD
  25. WHAT IS THE SIZE OF CYTOCHROME C IN kD?
    12.5 kD
  26. WHAT IS THE SIZE OF RIBONUCLEASE IN kD?
    12.6 kD
  27. WHAT IS THE SIZE OF LYSOZYME IN kD?
    13.9 kD
  28. WHAT IS THE SIZE OF MYOGLOBIN IN kD?
    16.9 kD
  29. WHAT IS THE SIZE OF HEMOGLOBIN IN kD?
    64.5
  30. WHAT IS THE SIZE OF AN Ab (IMMUNOGLOBULIN) IN kD?
    149 kD
  31. WHAT'S BIGGER IN SIZE A LYSOZYME OR A HEMOGLOBIN?
    HEMOGLOBIN

    • HEMOGLOBIN IS 64.5 kD
    • LYSOZYME IS 13.9 kD
  32. Gel permeation chromatography (GPC), Molecular Sieve Chromatography,
    Gel Filtration Chromatography (GFC)

    ALL SEPERATE BASED ON WHAT?
    Separation/purification based on size and shape through “bead” packing.
  33. DEFINE Relative elution volume IN SIZE EXCLUSION CHROMATOGRAPHY (SEC).
    • Ve/Vo
    • (helps to normalize between columns).
    • Vo= Void volume. VOLUME TO ELUDE A PROTEIN THAT DOES NOT RETAIN IN PACKING. 1L IN 1L OUT. EX: Blue dextran—very large dyed molecule that used as a standard.
    • Ve = Elution volume. FLUSH.
  34. DEFINE EXCLUSION LIMIT IN SIZE EXCLUSION CHROMATOGRAPHY (SEC).
    • BEYOND THE LIMIT NOT RETAINED IN PACKING.
    • EX: A BIG PROTEIN JUST FALLS THRU.
  35. IF ONE WANTS TO MEASURE MOLECULAR MASS OR TO DESALT PROTEIN SOLUTION WHAT CHROMATOGRAHY METHOD WOULD ONE USE?
    • SIZE SEPERATION CHROMATOGRAPHY.
    • EXAMPLES:
    • Gel permeation chromatography (GPC), Molecular Sieve Chromatography,
    • Gel Filtration Chromatography (GFC)
  36. THE VOLUME TO ELUDE A PROTEIN THAT DOESN'T RETAIN IN THE PACKING OF A SIZE EXCLUSION CHROMATOGRAPHIC METHOD IS CALLED WHAT?
    • VOID VOLUME
    • Vo
    • Volume for non-retained protein. ”too big to enter pores”
  37. THE VOLUME A PROTEIN SPENDS (RETAINS)IN THE PACKING OF A SIZE EXCLUSION CHROMATOGRAPHIC METHOD IS CALLED WHAT?
    • Ve= elution volume.
    • Volume protein spends
    • in beads (is retained)
  38. T OR F?
    A LARGE PROTEIN WOULD HAVE A SMALLER VOID VOLUME THAN A SMALLER PROTEIN DUE TO THE FACT THE LARGE PROTEIN WOULD JUST FALL THRU THE COLUMN?
    TRUE.
  39. AN EXAMPLE OF THE PACKING IN A SEC WAS GIVEN. NAME THE CHARACTERISTICS.
    • CARBOHYDRATE POLYMER BEADS.
    • AQUEOUS SPACES WITHIN THE BEADS.
  40. DEFINE Size Exclusion Chromatography Plot of Standards & NAME THE AXIS.
    • Plot of relative elution volume versus Log molecular weight gives a graph that we can use to determine MW.
  41. IN A Size Exclusion Chromatography Plot of Standards, WHERE WOULD ONE FIND A LARGER PROTEIN? LEFT TOP OR BOTTOM RIGHT?
    • BOTTOM RIGHT.
    • LOWEST AMOUNT OF VOID VOLUME ON CHART.
  42. HOW DOES ONE DETERMINE WHAT TYPE OF SIZE EXCLUSION GEL TO UTILIZE?
    • Pick based on size of interest
    • and number of proteins fractionated
  43. NAME 3 GELS AND SIZE RANGE UTILIZED IN SIZE EXCLUSION CHROMATOGRAPHY (SEC).
  44. WHAT IS THE Structure of Sephadex AND WHERE WOULD YOU FIND IT?
    USED AS PACKING IN A SIZE EXECULSION CHROMATOGRAPHY.

    IT'S A Cross linked carbohydrate matrix.

  45. DEFINE AFFINITY CHROMATOGRAPHY
    • LIGAND OR "MOLECULE HAND" BOUND
    • TO COLUMN PACKING/BEADS.
    • SOME "MOLECULAR HANDS" INCLUDE:
    • SUGARS, VITAMINS, ANTIBODIES & NTA/His TAGS
  46. NAME A CHROMATGRAPHTIC PROCESS THAT HAS EXCELLENT SEPARATION RESULTS TO TO SPECIFICITY, A 10,000-FOLD PURIFICATION.
    AFFINITY CHROMATOGRAPHY
  47. T OR F?
    ONE HAS TO HAVE THE LIGAND AND BE ABLE TO CONJUGATE IT IN ORDER TO EXECUTE AN AFFINITY CHROMATOGRAPHY.
    TRUE
  48. HOW DOES SIMPLE AFFINITY CHROMATOGRAPHY WORK?
    • Adding “free” glucose competes
    • with linked
    • glucose for glucose binding site on protein.

    • With binding (unless covalently attached) is in a constant state of “on
    • And “off” hence the ability to compete.
  49. DEFINE His TAGGING
    Expression or “engineered” protein production in bacteria or mammalian cells with histidines.

    Multiple histidines in a row at either the N or C terminus
  50. T OR F ?
    THE PROBLEM WITH His TAGGING IS THAT histidines have to be exposed on surface of protein and not buried in interior the core.
    TRUE
  51. IN His TAGGING, WHAT IS THE METAL INVOLED?
    • NICKLE.
  52. NAME THE RING FOUND ON HIS
    Imidazole
  53. WHAT DOES ONE ADD WHILST UTILIZING His-TAGGED AFFINITY CHROMATOGRAPHY?
    • •Add Lysate with His tagged
    • protein
    • •Wash
  54. IN His-tagged Affinity Chromatography, WHAT IS THE FUNCTION OF
    Imidazole?
    Add to remove protein OF INTEREST--completes with Histidine binding, PROTEIN OF INTEREST IS RELEASED.
  55. T OR F?

    IN FPLC (Fast Protein Liquid Chromatography) ONE CAN USE 2 BUFFERS IN ONE SYSTEM.
    • TRUE
  56. NAME 3 ANALYSIS METHODS THAT ARE USED THROUGHTOUT THE PURIFICATION PROCESS.
    • 1. GEL ELECTROPHORESIS...SDS/PAGE
    • 2. ELISA/WESTERN BLOT
    • MASS SPECTROMETRY....MALDI
  57. T OR F?
    ELECTROPHORESIS IS THE OPPOSITE OF RUNNING A COLUMN (SIZE EXCLUSION).
    • TRUE.
    • SMALL MOLECULES WILL RUN FIRST, LARGE ONES LATER IN A GEL.
  58. POLYACRYLAMIDE GEL IS USED TO RUN LARGE OR SMALL MOLECULES?
    • SMALL.
    • TO GET REALL SMALL INCREASE THE T%
  59. WHAT IS THE GEL ELECTROPHORESIS FORMULA?
    V = Ez / f

    • v = velocity of migration
    • E = electric field strength
    • z = net charge
    • f = frictional “drag”
  60. IN THE FORMULA OF GEL ELECTROPHORESIS, IF ONE HAD A LARGE f VALUE, WHAT WILL THE V VALUE BE?
    V WILL BE SMALLER BECUASE IF YOU HAVE A LARGE f (DRAG) VALUE THE V (VELOCITY OF MIGRATION) VALUE WILL BE SMALLER. AND OF COURSE, VISE VERSA.
  61. IN THE FORMULA V = Ez/f HOW DOES ONE CHANGE THE Z VALUE?
    Z VALUE IS THE NET CHARGE OF THE MOLECULE. ONE CAN ADD SDS TO NEUTRALIZE THE CHARGE, AND THUS THE VALUE IS CHANGED.
  62. IN GEL ELECTROPHORESIS SEPERATION IS BASED ON WHAT?
    Separation is a function of size/frictional drag.
  63. T OR F?
    IN THE FORMULA OF GEL ELECTROPHORESIS V = Ez/f THE E (ELECTRICAL FIELD STRENGTH) IS ALWAYS CONSTANT.
    TRUE.

    Most proteins bind ~1.4g SDS per g protein
  64. WHAT THE HELL DOES SDS-PAGE MEAN?
    Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
  65. IN SDS-PAGE, NAME THE 2 AGENTS THAT DENATURE PROTEINS.
    • SODIUM DODECYL SULFATE
    • MERCAPTOTHANOL (reducing agent).
  66. IN THE FORMULA OF GEL ELECTROPHORESIS V = Ez/f THE E (ELECTRICAL FIELD STRENGTH) IS TRANSFORMED INTO A CONSTANT. HOW?
    THE SDS Imparts a negative charge that obliterates the native charge of protein (gets a constant charge)

    Constant charge to mass ratio
  67. T OR F?

    Given that proteins have same mass to charge ratio and their native charge has been obliterated, larger proteins
    will experience more frictional drag and move more slowly through the gel.
    TRUE
  68. IN SDS-PAGE, THE MERCAPTOTHANOL (reducing agent) ACTS ON WHAT BOND TYPES.
    DISULFITE
  69. NAME THE AXIS ON A SDS-PAGE GRAPH.
    • PROTEIN MASS VS RELATIVE MOBILITY
    • LOG OF MW (kD) Y AXIS (UNEVEN TICKS)
    • MOBILITY (mm) X AXIS
  70. RUNNING A SDS-PAGE GEL? NAME 3 PURIFICATION PROCESSES.
    • ANION EXCHANGE
    • SIZE EXCLUSION
    • AFFINITY PURIFICATION
  71. T OR F ?
    IN AN SDS-PAGE RUN, ONE MAY DO MULTIPLE PURIFICATIONS TO GET CLEANER.
    TRUE
  72. Electrophoretic separation based on -----.
    Charge
  73. IN ISOELECTRIC FOCUSING, pH Gradient is established prior to the run by prefocusing ------- ---------.
    carrier ampholytes (CAs)
  74. WHAT THE HELL ARE carrier ampholytes (CAs)?
    small MW aliphatic compounds with an amino and carboxylic acid group (get a complete range of isoelectric points.
  75. Carrier ampholytes (CAs) are small MW ----- compounds with a ----- & ----- ----- groups.
    • aliphatic
    • amino and carboxylic acid
  76. WHAT THE HELL IS THIS AND WHAT DOES IT PERTAIN TO?
    • THIS IS WHERE ONE WOULD prefocus carrier ampholytes (CAs) small MW aliphatic compounds with an amino and carboxylic acid group. TO OBTAIN pI POINT VIA ALTERING pH.
  77. T OR F?
    IN ISOELECTRIC FOCUSING A BASIC PROTEIN WILL STAY TOWARDS THE CATHODE END?
  78. NAME TWO BUFFERS ONE CAN USE DOING ISOELECTRIC FOCUSING.
    NaOH: CATHODE, BASIC, NEG CHARGE, pI=8

    H3PO4: ANODE, ACIDIC, POS CHARGE, pI=4
  79. WHO IS 50,000kD @ pI 3.5?

    NUMBER 3

    • 1. 100,000 kD @ pI=8
    • 2. 1,000 kD @ pI = 3.5
    • 3. 50,000 kD @ pI = 6
  80. 2D-GE- Isoelectric Focusing + SDS-PAGE NAME STEPS FROM SLIDE.
    • Iso gel is equilibrated in SDS-PAGE buffer.
    • Gel is overlaid on top of an SDS-PAGE gradient gel.
    • Proteins separated on basis of MW.
  81. WHAT ARE THE 2 AXIS ON A 2D GE GEL RUN?
  82. WHAT 2 PIECS OF INFO DOES ONE OBTAIN FROM A 2D-GE- Isoelectric Focusing + SDS-PAGE?
    CHARGE AND MW
  83. T OR F?
    2D-GE- Isoelectric Focusing + SDS-PAGE IS OFTEN USED FOR LOOKING AT CELL LYSATE BEFORE PURIFICATION TO IDENTIFY PTROTEINS OF INTEREST.
    TRUE
  84. HOW DOES ONE IDENTIFY THE SPOTS OF A 2D-GE- Isoelectric Focusing + SDS-PAGE
    MASS SPEC
  85. WHAT IS USED to detect and quantify protein band on gel after running SDS-PAGE or 2D gel electrophoresis.
    • WESTERN BLOT (IMMUNOBLOT).
    • USED FOR PROTEIN CONFIRMATION.
  86. WHAT DOES ELISA STAND FOR?
    enzyme-linked immunosorbent assay (ELISA).
  87. NAME THE BASIC STEPS OF A WESTERN BLOT.
    • 1.preform a Electrophoretic Separation of Proteins.
    • 2. Transfer Proteins to a nitrocellulose membrane.
    • 3. Blocking Nonspecific/unoccupied binding Sites on the nitrocellulose membrane.Casein non fat milk.
    • 4. Incubate with Ab to the protein of interest. Binding of primary Ab. EX:Rabbit Ab.
    • 5. Wash & incubate with Secondary Ab.Binding of enzyme-linked secondary Ab. EX: enzyme-linked goat anti-rabbit Ab.
    • 6. Assay the linked enzyme with a colourmetric reaction. EX: HRP
  88. T OR F?
    IN A WESTERN BLOT YOU need antibody that is specifically generated and binds to protein of interest.
    TRUE
  89. FIRST 4 STEPS OF WESTERN BLOT.
  90. SET UP STEP 2 OF WESTERN BLOT. HOW?
  91. AT THE END OF A WESTERN BLOT WHAT DO YOU KNOW?
    • CONFIRMATION OF SIZE, CHARGE AND OBSERVED PRESENCE.
    • GO PUBLISH A PAPER.
  92. "Detect protein of Interest by Specificity in a plate". WHAT METHOD IS THIS?
    • ELISA.
    • Enzyme Linked Immunosorbant Assay.
    • USED TO CAPTURE Ab OUT OF SOLUTION.
    • COLOUR=VALUE
  93. NAME THE 4 STEPS OF ELISA
    • 1. IMMOBILIZE FIRST Ab ON SOLID SUPPORT.
    • 2. INCUBATE WITH PROTEIN CONTAINING SAMPLE.
    • 3. ADD A 2ND Ab THAT IS COVALENTLY LINKED TO AN ASSASABLE ENZYME.
    • 4. WASH AND ASSAY THE ENZYME.
  94. WHAT PROCEDURE?
    ELISA
  95. Clean Up Methods to get rid of small contaminants(used throughout process).
    NAME 3.
    • 1.Dialysis.
    • 2.Spin Filters (Centrifugal Concentrators) centrifugation with dialysis membrane.
    • 3.Desalting columns (SEC)-just like purification.
  96. WHAT DOES MALDI-MS STAND FOR?
    Matrix Assisted Laser Desorption Ionization Mass Spectrometry.
  97. BASICALLY HOW DOES Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) WORK?
    • LASER HITS PLATE.
    • PLATE TRANSFER E TO PROTEIN.
    • PROTEIN DISCHARGE UP TO ACCELERATOR TUBE.
    • MAGNETS PULL/DRIFT THRU TUBE TO DETECTOR.
  98. WHATS THE FIRST PART OF MALDI-MS?
  99. WHATS THE 2ND PART OF MALDI-MS?
  100. WHAT IS A MODERN TECINQUE FOR OBTAINING m/z (mass/charge) of a protein?
    MALDI-MS PROCESS.
  101. IF YOU RAN A GEL AND GOT A MW OF 100,00 FOR A PROTEIN AND THEN ADD REDUCING AGENT AND GOT A MW OF 50,000. WHAT THE FUCK HAPPENED?
    THE DISULFIDE BONDS BROKE AND NOW YOU HAVE 2 SUBUNITS OF THE PROTEIN.

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