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170 Cards in this Set

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