QBM Exam 2

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  1. What method can analyze both DNA and Protein sequences?
    BLAST Homolgy Searching
  2. What is the simplest method to synthesize a protein?
    Organic Chemistry
  3. What is the limitations to protein synthesis via Organic Chemistry?
    • Can only synthesize proteins ~50-70 amino acids in size
    • Proteins may not properly fold due to not being expressed in a cell
  4. What is a Vector (construct)?
    The bacterial plasmid to which a Human Gene is inserted into
  5. What is Recombinant DNA
    A vector containing DNA from various species
  6. Describe the in vitro (cell free) protein expression system
    • Simplest/most straightforward expression system
    • Only involves a ribosome and mRNA of interest
    • Fast
    • Produces a pure protein sample
    • Expensive and low yield
  7. Describe the protein expression system of Prokaryotic Cells
    • Easiest to work with as prokaryotic cells take up plasmid DNA easily and divide fast
    • Bacteria are cheap and easy to grow
    • High yield
    • Can express proteins that are toxic to eukaryotes
  8. What is the most well established and cost-effective system for protein expression?
    Prokaryotic (bacterial) Cell method
  9. Can the Prokaryotic Cell method for protein expression work for all proteins?
    No; some protein may not fold properly and form Inclusion Bodies (misfolded protein aggregates)
  10. Why would the use of Guanidine-HCL and Urea on a protein imply?
    Guanidine-HCL and Urea are harsh chemicals used in an attempt to refold proteins from inclusion bodies
  11. What is the temperature at which Bacteria grow best?
    37 celcius
  12. Describe the Yeast and Plant Cell method of protein expression
    • Use of eukaryotic systems
    • Can produce large (size) proteins
    • More likely to fold properly/contain proper modifications
    • Harder than use of bacteria 
    • Tedious and time consuming
  13. One of the most efficient animal cell systems uses _____ to infect ______.
    One of the most efficient animal cell systems uses Baculovirus to infect Insect Cells.
  14. Describe the protein expression system of using Baculovirus to infect Insect Cells
    • Large proteins (5000 amino acids)
    • High yield
    • Proper folding and modifications
    • Disulfide bond formation
    • Oligomerization
    • Simultaneous expression of multiple genes 
    • Not very straightforward, timely and costly
  15. Describe the use of Mammalian Cells for expressing proteins
    • Cells often used as a starting material for purification
    • Obtain large amounts of organs and purify proteins directly from them 
    • Not feasible for human proteins 
    • Final product is in it's native state, properly folded and modified
    • Hard to work with, low yield, expensive, time consuming 
    • Special media and incubator requirements
    • Not optimized yet
  16. Describe why the following special E. coli cell lines are used:

    • BSL21: carry T7 RNA polymerase gene
    • B834: methionine aucotrophs (can't synthesize methionine)
    • Turner: callow tighter control of expression
    • Mach1: grow very rapidly
  17. Describe the process of expressing proteins in Bacteria
    • Grow clone containing vector of interest at 37C in 100ml LB Media (lysogeny or luria broth) overnight in ~1mM Ampicillin (kills cells that don't carry the plasmid)
    • Transfer culture to 1L flask of LM media and ampicillin next morning
    • Allow to grow at OD600 (E. coli's exponential growth phase)
    • Induce cells to express protein via IPTG for 5hrs at 37C or up to 24hrs at 20C
  18. Why is it so important to control exactly when the protein of interest is expressed in bacteria?
    One needs to wait for bacteria to be at a high enough population density to produce ample amounts of protein, as expression of recombinant protein can halt/kill the bacteria
  19. The Lac Operon is used by bacteria to express proteins that break down ______.
    The Lac Operon is used by bacteria to express proteins that break down lactose.
  20. Describe the Lac Operon
    • It is a "system" used by bacteria to express proteins that break down lactose
    • In absence of lactose, lac operon is "off" and the lac repressor protein is bound to the operator, preventing RNA polymerase from transcribing the necessary mRNA required to break down lactose
    • Lac Operon is only "on" in presence of lactose, thus can easily be controlled to express recombinant proteins
  21. What is the Ptac Expression Vector
    • A simple expression system that makes use of the Lac Operon 
    • Example is the pGEX Vector
    • Has a Ptac promoter that is silenced by the lac repressor protein
    • Gene of interest is downstream of promoter and cannot be expressed 
    • System is "turned on" via addition of IPTG (lactose variant that can't be metabolized)
    • IPTG causes removal of lac repressor from operator allowing RNA polymerase to transcribe gene of interest
    • Ribosomes synthesize protein of interest

    Can be "leaky", low levels of protein expression even if system is off, can result in misfolded proteins
  22. What is the Phase Expression Vector
    • Example is the pET Vectot
    • Has tighter control than Ptac Vector
    • Uses the proteins of the host, bacteriophage, and lac operon
    • BSL21 bacteria carry bacteriophage T7 RNA Polymerase, upstream of which is the lac operon which is bound by the lac repressor (preventing protein expression)
    • IPTG turns system "on", lac repressor removed from promoter, RNA Polymerase and ribosomes transcribe T7 RNA polymerase
    • T7 RNA Polymerase targets T7 promoter on plasmid and transcribes gene of interest into mRNA
    • mRNA is translated to protein by ribosomes in high yield
  23. What is the purpose of adding PMSF, EDTA, or EGTA to a stored protein sample's buffer?
    They are Protease Inhibitors that aid in prevention of protein degradation
  24. What method might be interfered by the use of chemicals such as detergents or organic solvents which were used to lyse a bacteria?
    Bradford Method as it is not detergent compatible like the DC Protein Assay
  25. Give 3 examples of Enzymes used to cellular membranes
    • Lysozyme (for bacteria)
    • Cellulase (for plants)
    • Trypsin (for animals)
  26. What is the downside of using a French Press to lyse bacteria?
    • Lysate can be very viscous due to intact bacterial DNA
    • Can clog purifications columns
  27. How do you work around the "column clogging issue" that a French Press might cause wit your lysate?
    Use Sonication or Centrifugation to break p and remove the intact bacterial DNA and otter impurities prior to loading sample onto a column
  28. In what circumstance would you see a protein stored in 30% glycerol? Is there anything that must be done to the sample when you want to use it?
    • 30% glycerol is added to a protein sample that is being frozen and stored
    • Must be removed via Dialysis
  29. Why is Precipitation of protein sample avoided?
    It is a state of aggregated, denatured proteins (which are useless)
  30. What is an Ammonium Sulfate Cut?
    • Controlled method of protein precipitation
    • Ammonium Sulfate is a Structure Forming Salt 
    • Proteins are gently removed from buffer and stabilized by the (+) charges of Ammonia and (-) charges of Sulfate
    • Does not require water present in sample
    • Can be used in a gradient with increasing fractions to find protein of interest
  31. What method can lead to isolation of new proteins?
    Ammonium Sulfate Cut
  32. What method would you use if chromatography was not successful or if you want to clean up a lysate prior to adding it to a column? Why?
    • Ammonium Sulfate Cut
    • Allows protein purification while keeping proteins in native structure and with appropriate modifications
  33. What is the major disadvantage of Ammonium Sulfate Cuts
    • Typically a low yield
    • Proteins may aggregate with their natural binding partners, making it unable to be used by Affinity Chromatography
  34. What wave length does a Chromatogram read at?
  35. What is the purpose of the Solvent Precipitation Method?
    • Method for purifying proteins
    • Used to denature, inactive, or remove aggregate proteins
    • Phenol or Chloroform used when proteins can withstand harsh chemicals
    • May render proteins unable to be used in downstream techniques
    • Usually reserved for DNA and virus purificaitons
  36. Proteins are least soluble and often precipitate when ________. (Is this always a bad thing?)
    • Proteins are least soluble and often precipitate when the pH of the solution equals their pI.
    • Can be exploited to precipitate protein of interest or aggregates; most useful when pI's are different
  37. After dialyzing a protein fro one pH to another, a precipitate of unwanted proteins formed. How and why would you remedy this?
    Precipitate of unwanted proteins can be removed via centrifugation to improve protein purity
  38. What are Inclusion Bodies, and where are they typically found?
    • Improperly folded insoluble protein aggregates
    • Typically found in the pellet (insoluble) fraction
  39. How can you attempt to remedy the onset of Inclusion Bodies?
    • Use Chaotropic Salts such as Urea or Guanidine-HCL which will decrease the tertiary structure of a protein, linearizing it.
    • Chaotropic molecules can then be slowly removed with a gradient on a column or by dialysis
    • Denatured proteins will hopefully refold
  40. When would you see the use of detergents such as Triton X-100, Sarkosyl, and CHAPS?
    • These detergents are used to re-solubilize aa protein found in an insoluble pellet
    • Useful when working with membrane proteins which tend to aggregate due to exposed hydrophobic regions that inserts into the cell's phospholipid bilayer
  41. What detergents could you use to remedy a situation where you find your protein of interest is found within an insoluble pellet?
    You could use the detergents Triton X-100, Sarkosyl, or CHAPS
  42. What is Dialysis (or Buffer Exchange) and why would you use it?
    • Dialysis/Buffer Exchange is a process in which proteins are placed in special membraneous tubings with very small holes that only allow buffers to pass through so that salts and other unwanted molecules can be removed from a sample while proteins can be transferred into the new buffer of choice
    • Used for further purification/concentration of protein samples after initial identification by other methods
    • Can also purify large proteins from small proteins
  43. What are the methods you can use to remove salt from your sample?
    • Dialysis
    • Size Exclusion Chromatography
    • A desalting column
  44. How might you separate large proteins from small proteins via Dialysis?
    Choose dialysis tubing with a Mw cutoff between the larger and smaller proteins, thus only allowing the smaller proteins to pass through the membrane
  45. Can Dialysis be used to test if two proteins interact?
    Yes; If two proteins interact, their Mw's combine. By choosing a Mw cutoff that is between the largest protein's Mw and the combined Mw you can test to see if there is any change in filtration
  46. What must one be careful of when undergoing protein purification with very high protein concentrations? Can the issue be remedied?
    • When approaching very high protein concentrations, a protein sample may turn yellow 
    • May precipitate 
    • Precipitation can be reversed by increasing salt concentration, which helps stabilize the protein
  47. What is the process of Lyophilization?
    • Method of protein concentration where the samples are dried under a vacuum over several days to a fluffy powder
    • Samples can then be stored for years in a freezer
  48. What is the technique of Ultrafiltration?
    • Method of protein concentration where proteins are placed onto a membrane that allows only buffer to pass through 
    • Done via use of Centrifuge Tubes or Nitrogen Pressure into a stirred cell
  49. What is the process of Reverse Dialysis?
    • Protein concentration method 
    • Place protein sample in dialysis tubing and surround it in Polyethylene Glycol to draw out the water and reduce buffer volume
  50. When and why would you see the use of Polyethylene Glycol?
    Can be used during protein concentration via dialysis; draws out water and reduces buffer volume
  51. Why and when would you see the use of the following:

    B-mercaptoethanol (B-ME)
    Dithiothreitol (DTT)
    Sodium Azide
    Ethylene Glycol
    • EDTA: chelate metals and inhibit proteases
    • B-ME and DTT: keep a reducing environment to prevent oxidation of Cysteines
    • Sodium Azide: antimicrobial properties
    • Glycerol and Ehtylene Glycol: cryoprotectants (prevent degradation while frozen)
  52. Who is credited with discovering/inventing the method of Chromatography?
    • Mikhail Tsvet
    • Russian-Italian botanists studying chlorophyll
  53. What is Thin Layer Chromatography used for? What is it's Stationary and Mobile phase?
    • Allows one to separate, analyze, and check the purity of organic compounds such as fatty acids, pesticides, and drugs
    • Stationary = Silica or Cellulose
    • Mobile = Solvent mixed with sample
  54. What are Gas, Liquid, and Thin Layer Chromatography generally not used for?
    The study or purification of DNA or proteins
  55. What is High Performance Liquid Chromatography (HPLC)?
    • Protein purification method that uses high pressure pumps (100 megapascals) to increase separation of a sample
    • Allows one to separate, identify, and analyze the compounds in a sample with high resolution
  56. What do Fast Protein Liquid Chromatography (FPLC) and Low Pressure Liquid Chromatography (LPLC) use in their columns, and what to they analyze?
    Use starch columns to analyze amino acids and proteins
  57. What is the major disadvantage of SEC?
    Sample must be loaded into top of column in very small increments (< 5% total column volume) to minimize Band Broadening (sample volume expansion due to turbulence)
  58. SEC contains small beads made up of _____.
    SEC contains small beads made up of cross-linked Dextran.
  59. Why are FPLCs preferred over LPLCs for protein purification?
    • In columns sometimes proteins come off at similar times, this is countered by using longer columns and smaller beads which will increase column pressure
    • FPLCs are designed for higher pressure and thus withstand this effect better than LPLCs
  60. What are the uses of SEC?
    • Separate proteins of different Mw from a lysate
    • Separate monomers from various oligomers
    • Separate proteins aggregates or degradations products from sample
    • Buffer exchange
    • Generate a Standard Curve (when using proteins with known Mw)
    • Test protein-protein interactions
  61. What is the requirement of proteins for SEC to be viable?
    Proteins must differ at least two-fold in Mw to be separated via SEC
  62. As pH increases above a protein's pI, _______. As pH decreases below a protein's pI, _______.
    • As pH increases above a protein's pI, protons disassociate from the protein resulting in a net (-) charge 
    • As pH decreases below a protein's pI, protons associate with the protein resulting in a net (+) charge
  63. In Anion Exchange Chromatography, a buffer must have a pH ______ than the pI of a protein to ensure _______. In Cation Exchange Chromatography, a buffer must have a pH ______ than the pI of a protein to ensure _______.
    • In Anion Exchange Chromatography, a buffer must have a pH higher than the pI of a protein to ensure the protein has a net (-) charge
    • In Cation Exchange Chromatography, a buffer must have a pH lower than the pI of a protein to ensure the protein has a net (+) charge.
  64. If a protein has a pH = 6.25, what buffer pH do you need for Anion or Cation Exchange Chromatography?
    • Anion - buffer pH must be 7.25 or greater
    • Cation - buffer pH must be 5.25 or lower
  65. Describe Hydrophobic Interaction Chromatography (HIC)
    • Separates proteins based on lack of charge
    • Used when there are available hydrophobic amino acids on a protein's surface (usually at the core away from water, occasionally can be found at surface)
    • Stationary Phase has hydrophobic functional groups
    • Salt Gradient starts off very high (highly polar) to elute non-binding proteins, then slowly reduces (making buffer more nonpolar)
  66. What is Reverse Phase Chromatography?
    • Similar to HIC, separates based on lack of charge
    • Used on small peptides
    • Peptides are denatured and loaded onto the column which contains long-chain hydrocarbons (C8-C18)
    • Peptides eluted with Acetonitrile or Methanol in order of increasing hydrophobicity
  67. What type of interactions does Affinity Chromatography use?
    • Usually involves weak interactions (hydrogen bonding, ionic interactions, hydrophobic interactions, van der waals)
    • Some new ones can use covalent bonding
  68. What is Gene Fusion Technique?
    • A type of Affinity Chromatography
    • The gene encoding the protein of interest is fused to a second gene encoding a protein (the Tag) that is easily purified by Affinity Chromatography
  69. What is the vector used for cloning in Gene Fusion Technique?
    A bacterial Plasmid (contains the "Tag")
  70. How is the recombinant tag sequence gene inserted into the plasmid in Gene Fusion Technique?
    Restriction enzymes and DNA Ligase
  71. In Gene Fusion Technique, how are the Tag sequence and gene sequence for the protein of interest separated?
    • The two proteins are separated by a Linker of amino acids
    • Prevents them from directly containing one another so that each protein folds properly and is active
    • Provides a protease cleavage site used to separate tag from protein of interest
  72. What are some various types of gene fusions and Tags?
    • GST
    • Maltose-binding protein (MBP)
    • Calmodulin-biding protein (CBP)
    • Chitin
    • Streptavidin
    • FLAG
  73. What is a major disadvantage of Gene Fusion Technique?
    • Requires cleaving the tag from protein of interest with a protease
    • Can be difficult, time consuming, sometimes cleave wrong target
    • Sometimes low yield
    • Protein may precipitate
  74. Some proteins use ______ to bind to metals
    Some proteins use histidine to bind to metals
  75. What is Metal-Chelate Chromatography?
    • A type of Affinity Chromatography
    • Nickel or Cobalt are loaded onto a column and proteins with histidine (his) tags are passed over the column
  76. What is typically done to the gene of interest in Metal-Chelate Chromatography?
    The gene of interest is genetically modified to encode a polyhistidine tag, usually 6-8 histidines in length at either the N- or C- terminus of the protein
  77. How are target proteins eluted in Metal-Chelate Chromatography?
    • Use of an Imidazole Gradient
    • Imidazole is the aromatic ring of a histidine, thus will compete with histidine for the bound proteins
    • Often followed up with IEX or SEC to get better purification though
  78. What is special about the use of a HaloTag in Affinity Chromatography
    • HaloTag (Promega) is the only Affinity Chromatography technique to make use of Covalent Bonds
    • Provides the best purifications, can be washed indefinitely and eluted when one wishes by a protease 
    • Protease (has a his-tag) can be removed from the solution by passing over a Nickel Column
  79. How does Native Gel Electrophoresis separate proteins?
    Separation based on both Size and Charge
  80. In an SDS-PAGE, the first few lanes will typically show _____.
    In an SDS-PAGE, the first few lanes will typically show what was loaded, the flowthrough, and wash (where contaminants are found), with the proteins of interest eluted later when the gradient reached sufficient concentrations to remove it from the column
  81. How does SDS-PAGE remove charge as a factor?
    • SDS binds to amino acids, coating the protein in an overwhelming negative charge
    • Along with heat (which breaks weak bonds), SDS will unfold and linearize the protein due to repulsion
  82. SDS binds to proteins in proportion to the number of ______, approximately one SDS per _______.
    • SDS binds to proteins in proportion to the number of amino acids, approximately one SDS per two amino acids 
    • Or ~1.3g SDS per 1g protein
  83. The Charge-to-Mass (SDS:amino acid) ratio of proteins will be essentially _______.
    The Charge-to-Mass (SDS:amino acid) ratio of proteins will be essentially identical for all proteins because SDS coating dominates the native charge of the protein.
  84. What type of buffer is added to an SDS-PAGE?
    Sample Loading Buffer (Laemmli buffer)

    • SDS: coats protein in negative charge
    • B-mercapthanol: breaks SS bonds
    • bromophenol blue: color tracker 
    • Glycerol: increases protein density
    • Loading Control: Can be used for normalization (example, B-actin)
  85. Polyacrylamide pore size is determined by _____.
    Polyacrylamide pore size is determined by the concentration of acrylamide.
  86. The higher the concentration of acrylamide, the _____ the pore size. The lower the concentration of acrylamide, the _____ the pore size.
    The higher the concentration of acrylamide, the smaller the pore size. The lower the concentration of acrylamide, the larger the pore size.
  87. If you are analyzing a protein with a small Mw, you will want ______ percentage of acrylamide.
    If you are analyzing a protein with a small Mw, you will want a higher percentage of acrylamide.
  88. What is the purpose of a Gradient Gel with acrylamide?
    • Acrylamide concentration increases towards the bottom of the gel, thus porocity decreases 
    • Can separate proteins with a wide range of Mw
  89. The Stacking Gel contains ___% acrylamide, while the Resolving Gel contains ___% acrylamide.
    The Stacking Gel contains 5% acrylamide, whole the Resolving Gel contains 15% acrylamide.
  90. The Stacking Gel contains a buffer with a pH of 6.8, whole the Resolving Gel contains a buffer with a pH of 8.3.
  91. What is the purpose of the pH difference between the Stacking and Resolving Gel?
    Creates an ion gradient via glycine and Cl- which carry the current and assist in the stacking process
  92. What is the technique of Silver Staining?
    • Method for protein visualization
    • 10-100X more sensitive than coomassie
    • Silver atoms bind tightly to proteins, produce black/purple bands
    • Can't be used for downstream techniques
  93. What is a good alternative to binding dye techniques for protein visualization?
    • Fluoresense
    • 2,2,2-trichoroethanol (TCE) reacts with tryptophan under UV
    • Can be used for downstream
  94. What is Relative Mobility (Rf)?
    • The ratio of the distance each protein migrates to that of the tracking dye (which has a high charge-to-mass ration and therefore migrates faster)
    • Used for more accurate Mw determination
    • Can be used in conjunction with a standard curve
  95. What are the applications of the SDS-PAGE?
    • Determine protein's Mw
    • Part of a purification method
    • Track expression and location of protein of interest 
    • Verify chromatogram
    • Used in conjunction with protein modification or mutation studies
  96. What does use of Native Gel Electrophoresis entail?
    Allows one to run proteins on a gel without denaturing them, therefore retaining their native structure and activity
  97. The buffer used in native gel is usually around pH ___, so most proteins will carry a ___ charge and migrate towards the _____ electrode.
    The buffer used in native gel is usually around pH 9, so most proteins will carry a (-) charge and migrate towards the positively-charge electrode.
  98. What is the major disadvantage of Native Gel?
    Cannot determine Mw, as a large and small protein can have a similar reading due to difference charges

    • Large+Low(-) = high up
    • Large+High(-) or Small+Low(-) = medium
    • Small+High(-) = low
  99. Native Gels can be used to analyze the degree of _____ and ______.
    Native Gels can be used to analyze the degree of phosphorylation and glycosylation.
  100. What major advantage does Native Gel hold over SDS-PAGE?
    • Can determine a protein's oligomeric state
    • SDS-PAGE would split a heterodimer into two bands
    • Native Gel would keep it as a single band

    All samples migrate as single bands on Native Gel regardless of oligomeric state
  101. Native Gels are especially useful for studying _____, and are often used in conjunction with SDS-PAGE to study this.
    Native Gels are especially useful for studying protein-to-protein interactions, and are often used in conjunction with SDS-PAGE to study this.
  102. What type of Native Gel does allow for Mw determination?
    • Pore-Limited Gel Electrophoresis
    • No SDS present
    • Mw can only be estimated, thus need to perform MassSpec
  103. What is the technique of Isoelectric Focusing (IEF)?
    • Allows for separation of proteins based solely on charge
    • Protein is immobile in electric field that has a net neutral charge, occurs when buffer pH = pI
    • Thus, separation based on pI value and useful for determining pI of a protein
  104. What is Two-Dimensional (2D) Gel Electrophoresis?
    • Combination of Isoelectric Focusing (IEF) and SDS-PAGE
    • IEF gel run and placed horizontally across stacking gel
    • Proteins are separated by pI in IEF, and then separate by Mw in SDS-PAGE
  105. How can you improve the resolution of 2D Gel Electrophoresis?
    Run a Native Gel followed by IEF and SDS-PAGE, a technique called 3D Gel Electrophoresis 
  106. What is a Western Blot?
    A technique used that allows one to detect and quantify proteins that react with specific antibodies which only react with the target protein
  107. What is an Antibody?
    A protein that binds to a specific sequence of amino acids, or Epitope
  108. What are Polyclonal Antibodies?
    • Antibodies produce by injecting foreign proteins into an animal
    • Specific to multiple epitopes on a protein
    • May cross-react with other proteins, thus high affinity but low specificity
  109. Antibodies are produced from ____.
    Antibodies are produced from B-cells in the blood or lymphatic system.
  110. B-cells can be isolated and fused with cancer cells to make ______, which produce _____.
    B-cells can be isolated and fused with cancer cells to make Hybridomas, which produce only a single antibody called a Monoclonal Antibody (which only targets a single epitope).
  111. What must be done prior to a Western Blot?
    • An SDS-PAGE must be run
    • Afterwards the gel is sandwiched and plaved flat onto a Nitrocellulose or PVDF Membrane
    • The membrane is then placed between two places of filter paper soaked in buffer (allows electric current to pass through)
    • Electric current perpendicular to gel causes proteins to migrate out of the gel onto membrane and adhere
    • This process is called The Western Transfer
  112. What would happen if an antibody was added to the membrane of a Western Transfer after protein adherence? How is this remedied?
    • The antibody would bing all over, as there are many free binding sites available
    • This is remedied by Blocking the membrane by placing it in milk, which ensures that all of the free protein-binding sites on the membrane are no longer available
    • This ensures the antibody binds to only the protein of interest
  113. What is done after the Blocking step of a Western Blot?
    The membrane is soaked in a solution containing a Primary Antibody to the protein of interest (can be monoclonal or polyclonal)
  114. What is something to note about a protein that has run through an SDS-PAGE while performing Western Blot?
    SDS-PAGE has likely denatured the protein, thus antibodies used must be specific for the denatures protein
  115. The best antibodies are to _____.
    The best antibodies are to linear conformational epitopes.
  116. How are unbound antibodies removed during a Western Blot?
    They are rinsed off with TBS-T, a detergent used to break weak interactions
  117. How is the presence of an antibody detected in Western Blow?
    • Addition of a Secondary Antibody which will react with any antibody of the same biological source as the primary antibody
    • Accomplished by injecting a Constant Region (Fc) of an antibody into another species 
    • Secondary Antibody can be covalently coupled to an enzyme that catalyzes chromogenic reactions, such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase.
    • Presence of secondary antibody determined by immersing membrane into substrate of the coupled enzyme, causing color change/fluorescence
  118. Do you see Primary Antibodies containing conjugated enzymes for detection often? Give an example.
    • No, it is rare as the process for generating them is very involving
    • Usually only seen in cases of very common antibodies such as Anti-His or Anti-FLAG antibodies
  119. What are the advantages of using the Primary/Secondary Antibody System?
    • Can target any antibody from primary source, allowing a single secondary enzyme to target all primary antibodies from another species
    • New discovered proteins can be used directly in a Western Blot if a conjugated secondary antibody already exists
    • Allows for multiple primary antibodies against multiple proteins to be used on a membrane, thus only a single secondary antibody needed to visualize all primary antibodies
  120. How can antibodies be detected?
    • Chromogenic Reaction
    • Autoradiography
    • Chemiluminesence 
    • Direct Infrared Fluorescence
  121. What is the technique of ELISA?
    • A technique that allows for the detection of an antigen of antibody in a sample
    • Does not include running an SDS-PAGE
    • Samples are placed in small wells and analyzed
    • If a reaction occurs, causes color change, indicating an antibody has bound to an antigen
  122. What is the major disadvantage of the ELISA technique?
    • Gives no information about Mw
    • Antibody in an ELISA could bind nonspecifically and produce a false positive
  123. What is the Indirect ELISA setup?
    • Identical to Western Blot in that the antigen is bound to the membrane, and a primary antibody is added to the well followed by a secondary antibody
    • Solution changes color when substrate added to the well
  124. What is the Sandwich ELISA setup?
    • Uses an antibody0coated membrane that binds to an antigen
    • Secondary antibody coupled to an enzyme binds to the antigen at second location
    • More specific, will reduce false positives as only the antigen of interest should remain in well after washing
    • Requires multiple antibodies that recognize the target antigen, not always available
  125. True or False: Antibodies can be used in purification of a protein
  126. What is Immunoprecipitation (IP)?
    • Protein purification method using antibodies
    • Involves precipitating an antigen out of a solution using an antibody as a purification step
  127. What is Co-Immunoprecipitation (Co-IP, or pull-down)?
    • Protein purification method using antibodies
    • Involves binding an antibody to a protein on solution, hoping to pull down other proteins in the complex with it to detect new protein-protein interactions
  128. What is Edman Degredation?
    • One of the first techniques developed for Protein Sequencing 
    • Involves N-terminal sequencing of a very pure protein sample
    • Can be cut directly from a gel or blot
    • Handled carefully, Keratin from skin can contaminant easily 
    • Limited to ~50-60 amino acids, generally first 10 are all that is needed
  129. What can a researcher perform if he is looking for a specific protein but the protein sequence is unknown?
    Enter it into a BLAST search
  130. What is Mass Spectrometry Sequencing?
    • A protein is digested, and the Mw is analyzed and compared to fragments
    • Protein sequence determined by overlapping the fragments
  131. What is Protease Degredation?
    A technique that allows for protein analysis
  132. What is a Protease?
    • An enzyme that cleaves a protein at a specific amino acid sequence
    • Can be used to analyze protein interactions also
  133. How can protein interactions be analyzed (what methods)?
    • Native Gel Electrophoresis
    • Co-Immunoprecipitation
    • Pull-Down
  134. What is the Far-Western Blot?
    • A variation of the Western Blot
    • Allows one to identify protein-protein interactions
    • A Native Gel (instead of an SDS-PAGE) 
    • Rabiolabeled bait protein probe (instead of a primary/secondary conjugation)
  135. What is the Yeast Two Hybrid System (or Bacterial Two Hybrid System)?
    • A very powerful technique for identifying new protein-protein interactions
    • Can screen a large library of potential partners

    System uses a reporter gene that is transcriptionally active, produces a color change in the yeast. The transcriptional activator binds to DNA and activates RNA polymerase. A library of binding partners is screened, if a partner Y interacts with X, the activating domain will interact with RNA polymerase, triggering reporter transcription
  136. What is calorimetry?
    • A technique for analyzing protein-protein interactions
    • Allows for the measurement of minute heat changes in a sample that indicates binding or conformational change
  137. What is Isothermal Titration Calorimetry
    • A technique for analyzing protein-protein interactions
    • Measures heat change upon binding when one protein is titrated into a tube containing a binding partner (such as a peptide and a protein that interact)
Card Set
QBM Exam 2
QBM Exam 2
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