chapter 5

Question 1

what are preparative purifications?

Answer 1

• intended to produce a significant quantity of purified proteins for subsequent use

Question 2

what are analytical purifications?

Answer 2

• produce a smaller amount of a protein intended for analytical purposes such as identification, quanitifcation, and functional studies

Question 3

what is the first step of protein purification?

Answer 3

• extract proteins and other cellular components from a biological sample. namely from cultured cells or from a tissue sample
  
  • unless the proteins are secreted by the cells into the surrounding solution, this must be done by mechanically lysing the cellular membranes, which can be accomplished in a variety of different ways
  • cell membranes can be lysed by repeated cycles of freezing and thawing, mechanical agitation, filtrating, treatment with organic solvents, or often with a detergent that disrupts the integrity of the membrane

Question 4

to protect against proteolytic degradation, the extraction mixture is often treated with?

Answer 4

• protease inhibitors and stored at lower temperatures outside the optimal range for enzyme function

Question 5

the next step pf extraction is to separate the proteins from other cellular components by?

Answer 5

• centrifugation
  
  • which segregates particles in solution by mass and density
    
    • heavier/dense on bottom
    • lighter on top

Question 6

another technique that is sometimes used to separate out proteins is to alter their solubility by changing the salt concentration of their surroundings:

Answer 6

• if salt is added to a solution, protein solubility will initially increase, as the salt ions block interactions between charged side groups, preventing aggregation. this increase in protein solubility is knonw as “salting in”. eventually if eneough salt is added, these ions will begin to compete with the charged side groups on proteins in their interactions with solvent molecules
• as less solvent becomes available to solvate protiens, protein solubility decreases “salting out”

Question 7

what is chromotography?

Answer 7

• refers to several techniques that separate components of a mixture

Question 8

what is the mobile and stationary phase of chromatography?

Answer 8

• the mobile phase carries the mixture through a solid stationary phase, with which the desired product interacts. the strength of these interactions results in differential retention of substances, meaning that substances that interact weakly with the stationary phase mill move through the stationary phase and elute out more quickly than substances that strongly interact with the stationary phase
• these interactions may depend on charge, polarity, pH

Question 9

what is paper chromatography?

Answer 9

• the stationary phase is a piece of filter paper and the mobile phase is a liquid solvent that carries the solutes in the sample up the filter paper via capillary action
  
  • a more common variation of this today is thin layer chromatography (TLC) which uses a glass or plastic sheet coated in a thin layer of an absorbent substance, like silica, instead of paper as the stationary phase

Question 10

TLC separates compounds by?

Answer 10

• polarity
  
  • silica is polar and that’s typically true of the stationary phase in TLC
  • the mobile phase is relatively non polar
  • “like dissolves like”
    
    • polar solutes will stick to the polar stationary phases, whereas more nonpolar solutes will travel upwards with the nonpolar mobile phase

Question 11

how far a solute moves up the stationary phase is a proxy measure of its polarity. this is approximated by?

Answer 11

• the retention factor Rf, which is equal to the distance a given solute has migrated up the stationary phase, divided by the maximum distance travelled by the mobile phase known as the solvent front
  
  • Rf = distance traveled / distance of solvent front

Question 12

this means that Rf can range from?

Answer 12

• 0-1
  
  • lower Rf calues are associated with more polar compounds

Question 13

what is used to visualize TLC results?

Answer 13

• UV light or iodine treatment

Question 14

what is column chromatography?

Answer 14

• the basic idea is that the stationary phase is not a rectangular sheet, but a column with a stockpot on the bottom that allows the content to be drained out. by successively draining the column into multiple receptacles, pure samples of teh various components in the mobile phase can be obtained
• the basic principle is the substance that interacts least with the stationary phase elutes out first

Question 15

what is high-performance liquid chromatography?

Answer 15

• the mobile phase is passed through a solid absorbent material under high pressure, which allows for a faster and more precise separation of the compounds in the mixture
• the solvent ysed as the mobile phase affects the electrostatic interactions between the sample and the stationary phase, so it’s important to choose the solvent wisely

Question 16

what is normal-phase HPLC

Answer 16

• the stationary phase is polar and the mobile phase is nonpolar
• this results in longer retention times for more polar compounds, both because they will interact more intensely with the polar statinary phase and because they are not optimally soluble in the nonpolar solvent comprising the mobile phase
• instead, more non-polar compounds will elute rapidly through the column: again, both because they will not want to interact very intensely with the polar starinary phase, and becasue they’re happy to be dissolved in the nonpolar mobile phase
  
  • reverse-phase HPLC reverses the polarity of the stationary and mobile phases, using a nonpolar stationary phase and a polar mobile phase which causes the polar molecules to elute first, while nonpolar molecules are retained by the column

Question 17

what is gas-liquid chromatography?

Answer 17

• the mobile phase is a gas, and the stationary phase is a liquid
• this is an ideal procedure for analyzing volatile compounds with boiling points up to 300-400ºC
• in gas chromatography, the sample is vaporized and mixed with carrier gases (He or H) that bubble through the liquid stationary phase, traveling at different rates based on differences in polarity and boiling point which correspond to differences in the ability of substances to interact with the liquid stationary phase
  
  • more specifically, compounds with low boiling points will vaporize first and elute more quickly through the column
  • furthermore, low boiling points tend to be associated with low polarity, so if the stationary phase is polar, then a nonpolar compound with a low boiling point will elute even mroe rapidly

Question 18

what is size-exclusion chromatography?

Answer 18

• captures small particles within a porous fel and allows larger particles to fall right through

Question 19

what is ion-exchange chromatogrpahy?

Answer 19

• selects for molecules with a certain charge
  
  • anion exchange- designed to attract negatively-charged amino acids
  • cation exchange- designed to attract positively charged amino acids

Question 20

what is affinity chromatography?

Answer 20

• takes advantage of the binding affinity of proteins for specific ligands by immobilizing a target proteins ligand throughout the stationary phase
  
  • as the sample passes through the column, the protein of interest binds non-covalently to its ligand while unwanted compounds elute through
    
    • subtype of this chromatography knwon as immunoaffinity chromatogrpahy embeds an antibody within the column resin to recover proteins bound specifically by that antibody

Question 21

what is electropheresis?

Answer 21

• an approach that separates molecules based on their migration in an electric field

Question 22

how does gel electropheresis work?

Answer 22

• samples are loaded into a polyacrylamide or agarose gel, and an electric field is generated that causes the molecules to migrate through the gel. this electric field causes the gel to have 2 poles, one at either end
  
  • positive pole (anode)
  • negative pole (cathode)
• in an electric field, negatively charged species will migrate towatds with positively-charged and positively charged species will migeate toward the negatively charged cathode
• type of electrolytic cell in which current is applied to drive an otherwise nonspon reaction
• the distance migrated depends on their charge (how strongly they are affected by the electric field) and their size (which affects how readily they move through the pores of the gel)
• pH of 9 and add anionic detergent like SDS

Question 23

what is SDS page?

Answer 23

• denatures proteins into their unfolded states but imparts an even distribution of negative charge per unit of protein mass
• disrupts noncovalent interactions between side chains

Question 24

how do we separate proteins by their charge property (pI)?

Answer 24

• isoelectric focusing
  
  • separates proteins based on their relative number of acidic and basic residues
• a special gel is used with a stable pH gradient. when an electric field is applied, a protein that is in a region of the gel where the pH is below itd pI will be positively charged and will migrate towards the negatively charged cathode until it reaches the region of the gel where the pH is equal to its pI. at this point, the protein will stop migration because its net charge is 0, and it will not experience any electric force. the opposite is true of a protein in a region of the fel where pH is greater than its pI: it will be negatively charged and will travel towards the positvely charged anode until reaching the region where the gel is equivalent to the proteins pI

Question 25

what is 2D electrophoresis?

Answer 25

• combines isolectric focusing with SDS page
  
  • first an electric potential is applied in one direction across a gel with a pH gradient to separate proteins by isoelectric point then the proteins are denatured with SDS and a new electric potential is applied perpendicualr to the first, separating proteins further by mass

Question 26

what is spectroscopy?

Answer 26

• can be used to quantify the amount of protein in a sample based on its absorption of light ata characteristic wavelength
  
  • in spectroscopy light is transmitted through a sample, and the amount of light transmitted through the sample is detected by the spectrometer. this is used to produce an absorbance spectrum with peaks of high absorption of light by the sample, corresponding to low transmittance of light through the sample
  • the relationship between solute concentration and its absoprtion of light is given by the Beer-lambert equation.

Question 27

what is the Beer-lambert equation?

Answer 27

• A = EcL
  
  • a is the solutrd absorbance
  • I is the transmitted intensity of a given wavelength of light
  • e is a constsnt known as the molar absorptivity of the solute at the wavelength of light transmitted
  • c is the concentration of the attenuating solute
  • L is the path length (cm)
    
    • higher levels of absorbance mean that more light is absorbed which means that mroe substance is presnt

Question 28

proteins absorb light most strongly in the UV range corresponding to?

Answer 28

• 200-400 nm, as aromatic side chains in proteins absorb UV light
• for bulk protein quantification, absorbance is usually at 280nm

Question 29

if we wish to visualize all the proteins in our sample, we could?

Answer 29

• stain the gel with a dye that binds indiscriminately to all proteins like Coomassie blue, which should reveal a distinct band for each protein at its expected position

Question 30

radioactive labelling of proteins can be used when?

Answer 30

• allow them to be visualzied by exposure to an X-ray film

Question 31

if the goal is to idnetify a specific protein or set of proteins, antibodies specific to those protiens can be applied to detect the species of interest. the antibody that binds directly to the protien, known as the primary antibody, may itself be tagged with a dye or fluorsecent marker, or more commonly a second antibody that recognizes and binds to the primary antibody will be tagged in order to visualize bands corressponding to the protein of interest. this is an immunochemical approach known as?

Answer 31

• Western blotting

Question 32

an important immunoassay is ?

Answer 32

• the enzyme-linked immunosorbent assay (ELISA)
• with ELISA the antigens in a sample are attached to a solid surface
• antibodies specific to the antigen of interest are then applied to the plate and these bind their partner antigens
• after washing to remove unbound particles, the amount of bound antibody is determined by an enzymatic reaction that produces a visible signal
  
  • the way this works is that the antibody is covalenetly linked to an enzyme and when the enzyme’s substrate is added to the reaction chamber, the enzyme catalyzes a reaction that produces a colour change, emission of fluorescence, or current that can be emasured to quanitfy the amount of protein antigen present in the original sample