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170 Cards in this Set
- Front
- Back
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Most common for seperating DNA fragments
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Agarose Gel electrophoresis
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T/F DNA is easier to seperate than proteins? explain.
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True, it is uniformly negatively charged
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What does the voltage strength depend on during agarose gel electrophoresis?
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The length of the gel and the potential difference at the ends
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Which end in agarose gel electrophoresis will DNA migrate to?
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The positive electrode (anode)
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What primarily determines DNA mobility in agarose gel electrophoresis?
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size of DNA fragments
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The velocity of migration in agarose gel electrophoresis is limited by what?
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the frictional force of the agarose gel matrix (i.e. pore size, or % gel used)
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What is the DNA size range for agarose gel electrophoresis?
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100 bp - 60 kb
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What is used for seperating smaller DNA fragments?
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Polyacrylamide gels
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What kind of polymer is agarose?
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linear
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What controls the density of agarose gel?
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the concentration
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tracking dyes such as _____ _____ are included to mark the position of DNA in agarose gel electrophoresis
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bromophenol blue
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What are the parameters that affect DNA migration in agarose gels?
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agarose concentration, applied voltage, and conformation of DNA
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DNA will migrate ______ proportional to the log of its molecular weight
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inversely
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The higher the voltage, the _______ larger DNA fragments migrate; use of lower voltages will make seperation more proportional based on DNA sizes and agarose %.
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faster
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______ _______ DNA will migrate faster due to superhelicity
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closed circular
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_______ _______ DNA will migrate somewhere in between depending on its helicity
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Nicked circular
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this DNA Migrates the slowest on agarose gel
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linear
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T/F Base composition and temperature generally are factors determing migration and resolution for DNA seperations.
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F
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Name the buffers used in agarose gel electrophoresis
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Tris acetate EDTA(TAE), Tris/borate/EDTA (TBE), and Sodium borate (SB)
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Which buffer has the lowest buffering capacity but provides the best resolution for larger DNA during agarose gel electrophoresis
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TAE
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SB buffer is good for what?
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Shorter analysis time; can use higher voltage
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DNA is typically visualized by staining with _____ _____.
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Ethidium bromide
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Suspected mutagen; stain for DNA
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Ethidium bromide
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___ ____ is used to visualize DNA stained with ethidium bromide.
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UV light
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T/F after agarose gel electrophoresis, DNA can be removed from the gel for further analysis
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T
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What is used for seperating large pieces of DNA (>20 kb)?
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Pulsed-field gel electrophoresis
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Invented to take advantage of a basic concept in chromatography
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HPLC
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In HPLC, what is done to increase the quantity of solid phase? explain what this leads to and what else is needed?
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The surface area of the adsorbent needs to be increased. This is done by reducing the size of the beads that make up the solid phase. However, this leads to very high back pressure and very slow flow rates. Add pressure!
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A "medium" pressure method of HPLC, the term is generalized due to popularity
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FPLC- fast protein liquid chromatography
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Why do biochemists use cloning techniques to incorporate a 6-histidine tag onto proteins?
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For purification purposes; can use affinity chromatography and use imidazole (something like the mobile phase) to elute
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What is the role of acrylamide during preparation of polyacrylamide gel?
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forms polymer for gel
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what is the role N,N'-methylene-bis(acrylamide) during preparation of a polyacrylamide gel?
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It reacts with the polymer to form crosslinks
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What is the role of tetramethylenediamine during preparation of a polyacrylamide gel?
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radical stabilizer
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What is the role of ammonium persulfate during preparation of a polyacrylamide gel?
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radical initiator
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What is the role and characteristics of the stacking gel in Discontinuous PAGE?
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Lower % acrylamide, smaller, lower pH
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What is the role of the resolving gel in discontinuous PAGE?
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higher % acrylamide, larger, higher pH
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How does addition of ammonium sulfate enhance binding of proteins to the stationary phase during hydrophobic interaction chromatography?
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Takes water away from surface of protein; reduce ammonium sulfate to reduce interaction
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describe the role of Tris HCl, 25 mM, pH 7.5 during protein purification.
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buffer
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role of EDTA, 1 MM in protein purif.
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chelator
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role of b-mercaptoethanol (BME) in protein purif.
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provides reducing environment; prevent disulfide bonds
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role of NaCl, 50 mM during protein purif.
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ionic stength for protein
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role of glycerol, 20% during protein purif.
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stabilize proteins
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How would glycerol be removed during protein purif.?
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Dialysis of protein
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Aninon exchange needs ______ amino acids and has a ________ stationary phase.
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negative, positive
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Cation exchange needs _______ amino acids and ________ stationary phase.
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positive, negative
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role of NaCl, 50 mM during protein purif.
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ionic stength for protein
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role of glycerol, 20% during protein purif.
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stabilize proteins
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How would glycerol be removed during protein purif.?
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Dialysis of protein
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Aninon exchange needs ______ amino acids and has a ________ stationary phase.
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negative, positive
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Cation exchange needs _______ amino acids and ________ negative stationary phase.
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positive, negative
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What do you elute the GST-fusion protein from the column with?
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free glutathione
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Affinity chromatography; surface bound w/ what groups?
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epoxy, aldehyde, or aryl ester
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Metal Interaction Chromatography is a form of what? What is the surface bound with?
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affinity chromatography, imidodiacetic acid + Ni, Zn, or Co
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What should be avoid during Metal Interaction chromatography?
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chelating agents
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What are the various types of electrophoresis?
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PAGE, SDS-PAGE, Tricine-SDS-PAGE, 2D Gel, and agarose gel electrophoresis
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Ohm's Law
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V=IR
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Where does the resistance come from during electrophoresis?
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Gel, solvent buffers, and samples
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Too much resistance in agarose gels will cause them to what?
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heat and then dissolve
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What are the components of acrylamide gels?
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acrylamide, N,N'-methylene-bis(acrylamide), TEMED, ammonium persulfate
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T/F polyacrylamide gels are made in water
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F - made in buffer
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what determines the pore size of polyacrylamide?
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% acrylamide
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PAGE stands for what?
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Polyacrylamide gel electrophoresis
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SDS does what?
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disrupts secondary and tertiary structures of proteins structures by breaking H-bonds and unfolding protein, prevents protein aggregation, "masks" charge on protein so that all proteins act the same as regards to charge, prevents protein shape from influencing gel run
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SDS-PAGE seperates proteins based on what?
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masses
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What is the purpose of discontinuous SDS-PAGE?
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to concentrate large volume samples in order to increase resolution
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What are the three components of Laemmli SDS Page (Discontinuous SDS-PAGE)
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stacking gel, resolving gel, and running buffer
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What the properties of the the stacking gel in discontinuous SDS-PAGE?
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pH 6.8 tris chloride, 3-4% acrylamide
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What are the properties of the resolving gel in discontinuous SDS-PAGE?
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pH 8.7, 5-12% acrylamide
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What are the properties of the running buffer in discontinuous SDS-PAGE?
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Tris chloride, 25 mM, pH 8.3, glycine, 192 mM
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glycine exists as a zwitterion at what pH?
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6.8
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What are some dyes used in for visualization in SDS-PAGE?
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commasie blue, spyro ruby red (fluorescent), silver stain
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What is the first step in 2D gel electrophoresis?
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Isoelectric focusing
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Organic compounds that have different pKa's. Used in 2D gel electrophoresis
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Ampholytes
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What does isoelectric focusing do?
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Seperates proteins based on their pI.
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What does the second step of 2D gel electrophoresis seperate proteins by?
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Molecular weight
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What are the "tools of the trade" for isolating enzymes?
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UV spectrometer, centrifuge, column chromatography equip. (columns, fraction collector pump (FPLC), Cell disrupter, Electrophoresis equipment
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Analytical level of enzyme purification; how much?
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<1 mg
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Preparative level of enzyme purification; how much?
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10 mg- 1 g
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Industrial level of enzyme purification; how much?
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1 g- 1 kg
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How is protein content measured?
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protein assays
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What are the two types of protein assays? Which one is more accurate?
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UV absorbance and Colorimetric assays, colorimetric assays are more accurate than UV
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Which assay is quick and non-destructive?
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UV absorbance
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What wavelength do W and Y absorb at?
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280 nm
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what wavelength do nucleic acids absorb at?
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260 nm
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what wavelength do peptide bonds absorb at?
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195 nm
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What are the types of Colorimetric assays?
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Biuret, Lowry, Bradford, BCA
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What does the Bradford Assay measure and what dye is it based on?
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Amount of protein, Coomassie Brillant Blue
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What is the first step in a colorimetric assay?
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take a series of standards and make a standard curve
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what proteins do you use for standards in a colorimetric assay?
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Pure beta-galactosidase or Bovine serum albumin
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What do you do after you add the dye in a colorimetric assay?
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graph amount of protein vs. optical density (O.D.) for the standards, take unknown and measure O.D.
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What is used for measuring enzyme activity?
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Spectrophotometric assay
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Enzyme-linked immunoassay (ELISA) requires what?
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an antibody to the protein of interest
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What process needs a specific antibody to the protein of interest?
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Western Blot
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Phenylmethylsulfonyl fluoride is a what?
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Protease inhibitor
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Name some techniques used in releasing protein from the cells.
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Lysozyme, Sonicator, French Press, Microfluidizer, High pressure homogenizer
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What can be used to exchange the buffer of the a protein solution?
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Dialysis
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Dialysis is a form of what?
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Size exclusion chromatography
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What is used to desalt and concentrate protein samples?
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dialysis
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What do you do with a solution of cells after you homogenate?
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Centrifuge and filter
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What is used to seperate cellular components?
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Differential centrifugation
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_____ ______ precipitation is a common technique to isolate protein.
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Ammonium sulfate
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It is called ______ when ions shield charges and allow proteins to fold.
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Salting in
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It is called ______ when ions compete with water to interact with side groups. When _____ is high enough, _____ wins causing protein to precipitate
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salting out, salt, salt
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Nonpolar, aliphatic amino acids.
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GAVLIMP
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Polar, uncharged amino acids
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TNCQS
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Positively charged amino acids
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HRK
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Negatively charged amino acids
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DE
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Aromatic amino acids
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YFW
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The net charge of any protein at any pH is determined by what?
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the amino acid composition
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Describe the components of column chromatagraphy and what does it accomplish?
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Mobile phase (generally liquid), Stationary phase (electrostatically charged ions bound to insoluble, chem. inert matrix), Elution of protein (with salt and alteration of pH). It seperates molecules based on charge.
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What is the generic protocol for chromatography?
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Prepare column, apply sample, wash, elute, analyze fractions
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HPLC
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High performance (pressure) liquid chromatography
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FPLC
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Fast protein liquid chromatography
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What are some different types of ion exchange substituents (attached to the matrix)?
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DEAE, TMAE, CM, SP, or S
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What seperates proteins based on differences in hydrophobicity?
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Hydrophobic interaction chromatography (HIC)
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What promotes hydrophobic interactions in HIC?
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High salt concentrations
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How do you elute an affinity chromatography run?
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introduce free ligand!
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Transmittance
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the ratio of intensities of transmitted and incident light
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A 100% value of T represents a totally ______ substance. A T value of ______ represents a totally opaque substance.
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transparent, 0
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Absorbance is proportional to both the _______ of the absorber and the thicknes of the layer. (Beer-Lamber Law)
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concentration
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What are the advantages of using radioisotopes?
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1. Sensitivity and simplification
2. Double labeling technique 3. Pulse-chase method (labeling tissues or cells) 4. Exchange analysis for measuring praticipation in reaction |
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What are the disadvantages of using radioisotopes?
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1. potential hazard
2. cost 3. disposal 4. liability |
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What are radioisotopes?
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Atoms with unstable nuclei that undergo transformation in other atms which have more stable nuclei. The transformation takes place by the release of energetic particles or radiant energy.
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What are isotopes?
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atoms of different masses but identical chemistry
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How many neutrons are in carbon-11?
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5
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How many neutrons are in carbon-13?
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7
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How many neutrons are in carbon-14?
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8
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what is released when a radioisotope converts a neutron to a proton?
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beta electron (negatron), different atom, and antineutrino
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What is released when a radioisotope converts a proton to a neutron?
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positron, different atom, and neutrino
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What are produced from the three different types of radioactive decay?
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alpha particles, beta particles (pos. and neg.), and gamma rays
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Type of particle never used in research, purely nuclear, low penetration ability.
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Alpha particles
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Used in research, medium penetration, negative and positive particles
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Beta particles
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Very high penetration, 1 type of particle, after the beta decay
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Gamma rays
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The mean energy is a characteristic of particular isotopes and be used to identify one _______ in the presence of another one.
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beta-emmiter
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Emission of ____-ray is often a secondary process occuring after the initial decay by ______-emmision.
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Gamma, Beta
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Beta particles are ejected from the nucleus with velocities approimately equal to the _____ __ _______.
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speed of light
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Beta particles are ejected with a ______ ______ of kinetic energies up to a maximum value, ____, which is characteristic of particular isotope.
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continuous range, Emax
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A plot of the relative probability of emission of a beta-particle as function of energy is called a _____-_____
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beta-spectrum
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When beta-particles pass through matter, their energy are dissipated by _______ and/or ________ of the atom with which they colloid.
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ionization, excitation
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Gamma rays have radioisotopes that emit one or more discrete energy values rather than over a ______ _____ as whith beta-particles
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continuous range
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A gamma-ray is ______ and therefore does not directly ionize atom in its path.
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uncharged
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The process of decay of radioisotopes nucleus is ________.
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irreverseible
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What are the common radioisotopes used in biochemistry? Which one is the only gamma radiation emmiter?
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3H, 14C, 32P, 35S, and 125I, 125I
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What are the units used in radioactivity measurement?
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eV, MeV, Bq, dps, cpm, mCi/mmole, Ci
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Bq=? which is a measurement of what?
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dps, activity
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dps stands for what? and dpm?
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disintegrations/second, disintegrations/minute
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disintegration rate per unit mass of radioactive atoms is know as what?
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specific activity
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cpm= dpm x ?
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E (efficiency)
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For measurement of ___ and ____ particles it is necessary to put the "detector" as close to the decay particles as possible
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alpha and beta
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For ___-___ detection, the detector may be outside the sample counter. So no cocktail is needed with ____ counter.
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gamma-ray, gamma
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What is used to measure beta activity?
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gas ionization
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what is a fluorescent substance know as?
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fluor
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The photon emitted by the fluor must be detected by a ?
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photomultiplier tube (PMT)
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What solvents are used in Liquid Scintillation Counting?
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Toluene, p-xylene, and 1,4-dioxane
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What fluors are commonly used?
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PPO, POPOP
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an emulsifier is a ____ type molecule
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detergent
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Regardless of the radioactive isotope, the wavelength of this scintillation light is dependent solely upon the ______. PPO normally generates light in the _______ (approximately 370 nm) region of the spectrum.
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fluor, blue
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The ___ ___ is proportional to energy of the particle coming (coming out of the PMT)
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pulse height
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what happens during scintillation counting?
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the emitted particles cause a pulse of light which is detected by an optical device (PMT) that converts it into an electrical pulse that can be counted
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A solution of ____ is called a scintillation cocktail.
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fluor
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What are some interfering processes during scintillation counting?
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Spurious generation of counts (chmilluminescence and photolluminescence) and Quenching of counts.
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Quench results in ?
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1. decreased number of photon/beta-particle
2. the production of a pulse of reduced voltage. |
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what are the three types of quench in liquid scintillation?
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chemical, color, and self-absorbtion quench
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What happens to the graph of two different beta-emitters during a quench effect?
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either a decrease in count or a shift of the curve.
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What two beta-emitters always show a shift on the graph during a quench effect?
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32P and 14C
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How do you get the actual sample count during quenching?
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take the sample cpm divided by the efficiency decimal number ( x cpm/ .xx)
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SQP stands for what?
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standard quench parameter
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cpm stands for what?
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counts per minute
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what method requires correction of the curve for quench correction?
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channels ratio method
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Beta-particlesilver bromidemetallic silver ; this happens during what?
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autoradiography
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