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Biochem > Lecture 3: Protein Purification and Characterization > Flashcards

Lecture 3: Protein Purification and Characterization Flashcards

1
Q

What must be done to study a protein of interest?

A
  • must be separated from all other cell components including other proteins
  • this is so structures and functions of the protein of interest can be probed without any confounding effects of contamination
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2
Q

Properties of proteins important in separation

A
  • solubility
  • size
  • charge
  • polarity
  • binding affinity

* has to be empirically optimized and specific for protein of interest

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3
Q

Charateristic: Solubility

Procedure?

A
  1. salting in
  2. salting out
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4
Q

Characteristic: Ionic charge

Procedures?

A
  1. ion exchange chromatography
  2. electrophoresis
  3. isoelectronic focusing
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5
Q

Characteristic: Polarity

Procedure?

A
  1. absorption chromatography
  2. paper chromatography
  3. reverse phase chromatography
  4. hydrophobic interaction chromatography
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6
Q

Characteristic: Molecular Size

Procedures?

A
  1. dialysis and ultrafiltration
  2. gel electrophoresis
  3. gel filtration chromatography
  4. ultracentrifugation
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7
Q

Characteristic: Binding Specificity

Procedures?

A
  1. affinity chromatography
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8
Q

Criteria to set before developing a purification process

A
  1. selection of protein source (cellular system - where are you looking for the protein?)
  2. methods of protein enrichment and solubilization (cell disruption, subcellular fracturation/centrifugation, membranre protein or soluble? )
  3. methods of protein stabilization (physiochemical conditions and protection from degradation, takes 2 days min to purify protein - how do you keep protein from degrading)
  4. protein assay or detection (enzymatic, antibody or other specific reactions - make sure the protein is in the sample)
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9
Q

Most common type of human protein?

A
  1. 2% enzymes
  2. 8% nucleic acids
  3. 4% unknown function!
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10
Q

Definition of centrifugation:

A
  • process that involves the use of centrifugal force for the sedimentation of mixtures with a centrifuge
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11
Q

Sedimentation

A
  • tendency for particles in suspension to settle out of the fluid in which they are entrained, and come to rest against a barrier

–> due to their motion through the fluid in resposne to forces (such as centrifugal acceleration) acting on them

  • differences in sediment properties of proetinous cellular components can be used for various purposes from subcellular fractionation to estimation of molecular masses
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12
Q

Centrifugal force equation

A

Fc = m (w2) r

m= effective mass of a sedimenting particle

w (omega) = angular velocity of rotation (radian/sec)

r = distance of the migrating particle from the center of the axis of rotation

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13
Q

Force on sedimenting particle (relationship to other variables)

A
  • the force on a sedimenting particle increases with:
  • velocity of rotation (w2)
  • effective mass of the sedimenting particle (m)
  • and distance from the axis rotation (r)

*directly proportional to all

Fc = m (w2) r

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14
Q

Buoyant force exerted by the solution

A

FB = v (p) m (w2) r

force on sedimenting particle is reduced by the buoyant force exerted by solution

  • v = the particles partial specific volume
  • the volume change when 1g of particles is dissolved in an infinite solute volume
  • for most proteins dissolved in water v = .74cm3/g
  • p = density of the solution
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15
Q

Overall force on sedimenting particle

“sedimentation force”

A

Fs = Fc - Fb = m(1-vp)w2r

v = particles specific volume = .74cm3/g for most proteins dissolved in water

p = desnity of solution

w = angular velocity

r = radius

m = mass of partile

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16
Q

Frictional force

A

Sedimentaion force is opposed by frictional force

Ff = (dx/dt)f

dx/dt = migration rate of sedimenting particle

f = frictional coefficient

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17
Q

Sedimentation force = frictional force

A

under influence of sedimentation force, the particle accelerates until the forces on it exactly balance

s = (dx/dt)/w2r = m(1-vp)/f

1 Svedberg unit (S) = 10-3 sec (as in 70s ribosomes)

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18
Q

Measuring sedimentation coefficient

A
  • can be measured by measuring the sedimenting particles at increasing time intervals

–> absorption sepctra for sedimenting proteins can be generated at a wavelength of 280 nm

  • a boundary is created that proceeds towards higher r values with increasing time, yielding a characteristic sedimentation profile

–> this boundary becoems less steep with time because diffusion breaks down the concentration gradient that is formed by sedimentation

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19
Q

Correlation of molecular Mass and sedimentation coefficient

(most important equation?)

A

M = (RTs)/(D[1-vp])

M = moleular mass (m X N; N = avogardos number)

R = gas constant (8.3145 J/(mol x K)

D= diffusion coefficient

v= particle’s partial specific volume

p = density of medium

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20
Q

Diffusion coefficient of a sedimenting particle

A

ds/dt = -DA(dc/dx)

ds/dt = amount of solute diffusing across area A in time t

dc/dx = concentration gradient

D = diffusion coefficient (solute diffusing across a surface area of 1cm2 per sec)

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21
Q

What happens in differential centrifugation?

A
  • a homogenate (mixture of disrupted cells) is centrifuged in a step by step fashion of increasing centrifugal force
  • forms supernatants and pellets with different densities
  • supernatants or pellets of each centrifugation step can be separated and further differentiated, depending on the cellular object of interest
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22
Q

Steps of tissue separation in differential centrifugation

A

–> tissue homogenate

–> whole cells, nuclei, cytoskeletons, plasma membranes

–> mitochondria, lysosomes, peroxisomes

–> microsomes (fragments of ER), small vesicles

–> ribosomes, large macromolecules

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23
Q

Preparative Centrifugation

A
  • sedimentation is carried out in homogenous media or density gradients (CsCl or sucrose) to separate proetinous components in homogenates or other samples

Two applications employed:

  1. Zonal Centrifugation: based on velocity sedimentation, separates particles according to molecular mass
  2. Isopycnic Centrifugation: based on equilibrium sedimentation, separates particles according to their densities
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24
Q

Zonal Centrifugation

A
  • separates according to molecular mass
  • separate by sedimentation coefficient

high density gradient, slows down centrifugation, gets bands

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25
Q

Ispoycnic Centrifugation

A
  • separates particles according to densities
  • based on equilibrium sedimentation
  • smaller particles move with less dense particles
  • larger molecules move with more dense particles
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26
Q

Meselson and Stahl Experiment

A
  • centrifugation to separate out different strands of DNA
  • Used 15N to ID original strand of DNA
  • when cells divided all new N was 14N
  • proved the semiconservative model

parental DNA –> 1st generation (2 strands, each one side had 15N) –> second generation (four strands - 2 half 14N/ half 15N, 2 all 14N)

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27
Q

Main conclusion of the Meselson and stahl Experiment

A

DNA replication in E Coli is semiconservative

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28
Q

Salting Out

A
  • describes the effect that at high ionic strengths the solubilities of proteins (and other substances) decrease, and proteins eventually precipitate
  • primarily a result of the competition between the added salt ions (ammoniumsulfate is most common) and other dissolved solutes for molecules of solvation
  • used to fractionate proteins (due to difference in precipitation points) or to concentrate diulted proteins
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29
Q

Why do proteins decrease solubility

A
  1. ionic interaction with H2O
  2. ionic interaction with protein itself –> precipitate
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30
Q

Salting out graph

A

proteins salt out below 0

31
Q

Dialysis

A
  • process that separates molecules according to size through the use of semipermeable membranes containing pores of less than macromolecular dimension

–> these pores allows small molecules (solvents, salts, small metabolites) to diffuse across the membranes but block the passage of larger molecules (depending on the molecular weight cutoff of the pores)

  • is useful for removing small molecules from a protein sample or for chanigng the buffer (rebuffer) conditions of the sample
32
Q

Dialysis and member Filter Devices

A
  • the removal of salts (or any microsolute) or the exchange of buffers can also be accomplished in memmbrane filter devices with defines pore sizes by first concentrating the sample and then reconstituting the concentrate to the original sample volume with any desired solvent
  • process of “washing out” can be repeated until the concentration of the contaminating microsolute has been sufficiently reduced
33
Q

Chromatographies

A
  • laboratory techniques used for the separation of mixtures, such as protein-containing solutions
  • take advantage of difference in charge, size, binding affinity and other properties of constituents within these mixtures
  • mixture is dissolved in a fluid called “mobile phase” which carries it through a structure holding another material alled “stationary phase”

–> various constituents of the mixture travel at different speeds, causing separation

34
Q

phases in chromatography

A
  • mobile phase: protein sample and solute
  • stationary phase: solid porous matrix
35
Q

Size Exclusion/Gel filtration chromatography

A
  • carbohydrate polymer beads (act like sponges)
  • small molecules enter the aqueous spaces within the beads (retarded)
  • large molecules cannot enter the beads (unretarded)

*large molecules move through faster

36
Q

Size exclusion chromatography: Total, bed elution and void volume

A

Vx = volume of beads

V0= void (or excluded volume)

Vt = total volume

Vt = Vx + V0

Ve = elution volume

Ve/V0 = relative elution volume

37
Q

Rates of protein travel in Size exclusion chromatography

A
  • proteins of different sizes penetrate into the pores of the beads to different degrees and travel down the column at different rates

–> very large proteins cannot enter the pores of the beads and thus remain excluded (V0) of the column - vlume of the aqueous phase outside the beads

–> small proteins, on the other hand, are retarded by the column

38
Q

Size exclusion chromatography: Elution volume

A
  • Ve is the volume of solvent required to elute the solute from th ecolumn
  • void volume V0 is the elution volume of a solute whose molecular mass is greater than the exclusion limit of a gel
  • behavoir of a particular solute on a given gel is Ve/V0
  • relative elution volume is soemwhat independent of the particular column used
39
Q

Size exclusion chromatography: void volume

A
  • measured as the elution volume of a solute whose molecular mass is greater than the exlusion limit of the gel
40
Q

Size exclusion chromatography: relative elution volume

A
  • Ve / V0
  • behavior of a particular solute on a given gel
  • quantity somewhat independednt of the particular column used
41
Q

Absorption spectra of eluted protein

A

use aromatic amino acids (PTT) at 280 to ID that a protein has been eluted

* other molecules are at other absorptions

  • carbs around 230
  • DNA/RNA around 260
42
Q

Ion exchange chromatography

A
  • positively charged proetin binds to negatively charged bead (or opposite)
  • negatively charged protein flows through
  • proteins move through the column at rates determined by their net charge at the pH being used
  • within cation exchangers, proteins with a more negative net charge move faster and elute earlier
43
Q

How do you elute the protein attached to the beads after ion exchange chomatography?

A

For a anion protein:

  1. change the pH of the buffer to below the IP (
  2. add salt that displaces/competes with the - proteins (ie Cl-)
44
Q

Ion Exchange chromatography and Acid/Base properties

A

manipulate pI and pH to get certain behaviors out of proteins

45
Q

Information from titration curve

A
  1. the quantitativ measure of thepKa of each of the two ionizing groups
  2. the regions of buffering
  3. relationship between the net charge and pH of the solution

–> pH where net charge is 0 is the isoelectronic point

–> for glycine, the pI is the arithmetic mean of the two pKA values

pI = 1/2 (pK1 + pK2)

46
Q

Isoelectronic point

A

relationship between the net charge and the pH of the solution

47
Q

Cation and anion exchange matrices

A

Cation exchange:

  • negatively charged beads. attract cations out of solution
  • protein has a high pI >7

Anion exchange:

  • positively charged beads. attract anions
  • protein has a low pI , 7
48
Q

Affinity chromatography

A
  1. protein mixture is added to column containing a polymer bound ligand specific for protein of interest
  2. unwanted proteins are washed through column
  3. Ligand solution is added
  4. protein of interest is eluted by ligand solution (competes out binding proteins to the beads)
49
Q

Affinity chrom: use of recombinant affinity tags

A
  • recombinant DNA techniques are used to make fusion between protein x and glutathione coated beads
  • when cell extraction is added, interacting proteins bind to protein x
  • glutathione solution elutes fusion protein together with proteins that interact with protein X
50
Q

Purification scheme of a hypothetical protein

A

1 crude cellular extract

  1. precipitation with ammonium sulfate
  2. ion exchange chromatography
  3. size exclusion chromatography
  4. affinity chromatography
51
Q

Electrophoresis

A

* have a purified protein, now must make sure it is purified

  • motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field and is widely used for analytical separation of biological macromolecules

–> only charged dispersed particles (ions) can migrate

  • common applications to characterize protein samples are

–> polyacrimide-based gel elecrtophoresis (1D and 2D)

–> isoelectronic focusing

52
Q

Forces on charged dispersed particles

(two)

A
  • electrical force Fe on an ion with charge q in an electric field of strength E

FE = qE

  • the motion of an ion through a solution due to FE is opposed by the frictional force, FF defined as

FF = (dx/dt)f

53
Q

Balances of forces on an ion

A

FE = qE = FF = (dx/dt)f

in a nonconducting solvent, each ion moves with a constant characteristic velocity

µ = (dx/dt) / E = q/f (in cm2 x V-1 x s-1)

54
Q

Direction of charged proteins in PAGE

A
  • negative charged will run cathode (-) to anode
  • positive will run anode (+) to cathode

*wont run opposite ways

55
Q

PAGE

A
  • polyacrylamide gel electrophoresis
  • separation of proteins accodring to their electrophoretic mobility
56
Q

Formation of polyacrylamide gels

(and resulting gels)

A
  • a three dimensional mesh is formed by copolymerizing activated monomer (acrylamide) and cross linkers (bisacrylamide)
  • polymerization is initiated by ammonium persulfate and tetramethylethylenediamine (TEMED)
  • more crosslinks = more dense gel will be less porous, only allow small molecules
  • fewer crosslinks = a more wobbly gel will be more porous and more gets through
57
Q

SDS PAGE

A
  • SDS is a detergent that unfolds and hence denatures proteins by disrupting non-covalent bonds
  • SDS binds (1.4 g/g) to proteins, forming negatively charged complexes that migrate in the direction of the anode (bottom of the gel)
    1. denature proteins
    2. add - charge (SDS)
    3. run cathode to anode

* proteins end up separating by size

58
Q

PAGE - reduction of disulfide bridges

A
  • a protein connected by disulfide bridges will end up in its pieces
  • if disulfide bridges are not reduced it will travel as one piece
59
Q

PAGE - Estimating relative molecular masses

A
60
Q

SDS PAGE - estimating relative molecular masses

A

X

61
Q

electrophoresis - isoelectric focusing

A
  • separation of proteins according to their isoelectric points
    1. an ampholyte solution is incorporated into a gel
    2. a stable pH gradient is established in the gel after application of an electric field
    3. protein solution is added and electric field reapplied
    4. after staining, proteins are shown to be distributed along pH gradient according to their pI values
62
Q

Isoelectric focusing and stable pH gradient

A
  • at low pH the protein is positively charged
  • at high pH the protein is negatively charged
  • at the isoelectric point the protein has no net charge and therfore no longer migrates in the electric field
63
Q

Two dimensional gel electrophoresis

A
  1. separation in first dimension by charge
  2. separation in second dimension by size

( because proteins can be different but have same MW or same charge –> separate by both!)

64
Q

Agarose gel electrophoresis

A
  • technical limitations to use polyacrymalide gels for separation of large molecula rmass compounds (above 200 kDa)

–> require large pores and hence gels with such low polyacrylamide concentrations (below 2.5%) that they are too soft to be stable

  • for these larger molecular mass compounds (up to 50,000 kDa) agarose gels are used
  • proteins or protein complexes
  • nucleic acids (DNA, RNA)
65
Q

What is agarose?

A
  • a mixture of sulfated heteropolysaccharides made up of repeating units of D-galactose and L-galactose derivative linked by an ether bond
66
Q

Agarose gel: restriction fragment analysis

(procedure)

A
  • DNA fragments produced by restriction enzyme digestion of a DNA moelcule are sorted by agarose gel electrophoresis

Procedure:

  1. prepare pure samples of individual fragments
  2. control DNA cloing experiments
  3. compare two very similar DNA molecules, such as two alleles for one gene (eg wild type vs disease) based on single nucleotide polymorphism (SNPs)
67
Q

Separation of DNA fragments obtained by digestion of a single DNA molecule with two different restriction enzymes

A
  • different restriction enzymes cut DNA in different places, yilding segments of different lengths
  • in response to an electrical charge, DNA fragments move through the gel
  • short DNA fragments move farther than longer fragments

if a gene is mutated and is missing a segment ir will fragment into a different number or pieces than normal

68
Q

Immunoblotting

A
  • proteins act as antigens
  • if an antibody to a protein of interest is available, it is possible to specifically detect this protein in the presence of many other proteins that have been separated via gel electrophoresis and transferred to a protein binding membrane (made out a material such as nitrocellulose)
69
Q

Immunoblotting - antibodies

A
  • antibodies - globular proeins called immunoglobulins
  • each contains a defined number (valence) of dentical sites that bind epitopes (antigen-binding sites)
  • the simplest structure is bivalent with four protein chains

–> two identical light chains and two identical heavy chains

70
Q

Immunoblotting - antigens

A

a substance that causes the body to produce specific antibodies or sensitized T cells

–> antibody production is a humoral response of the adaptive immune system

71
Q

What produces antibodies?

A

B lymphocytes (B cells)

72
Q

Immunoblotting procedure

A
  1. perform gel electrophoresis on a sample containing the protein of interest. Blot the proteins from the gel onto nitrocellulose
  2. block the unoccupied binding sites on the nitrocellulose with casein
  3. incubate with rabbit antibody to the protein of interest
  4. wash and incubate with an enzyme linked goat anti-rabbit antibody
  5. assay the linked enzyme with a colorimetric metric reaction

* use two antibodies and enzyme/substrate to AMPLIFY the signal

73
Q

Immunoblotting antigen detection

A
  1. coat surface with samples (antigens)
  2. blok unoccupied sites with nonspecific protein
  3. incubate with primary sntibody against specific antigen
  4. incubate with secondary antibody- enzyme complex that binds promary antibody
  5. add substrate
  6. formation of colored product indicates presence of sepcific antigen

Biochem (15 decks)

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