BB5&6 Protein Isolation & Purification

Question 0

The proteins in solution will denature unless they are stabilized against

Answer 0

• changes in pH (add buffer)
• changes in temperature (cool to 4c)
• protease degradation (add protease inhibitors)

Question 1

Protein Accessibility

Answer 1

• in solution
• in extra-cellular medium
• intra-cellular = must be broken (lysis)

Question 2

Protein separation uses differences in

Answer 2

• solubility
• size
• charge
• specific binding affinity

Question 3

After each step, a test ________ must be performed to see if the desired protein has been separated from the others

Answer 3

assay

Question 4

Salting out

Answer 4

• uses solubility differences
• different proteins salt out at different salt concentrations
• only partial separation

Question 5

Solubility changes with addition of

Answer 5

ionic salts

Question 6

Increasing salt concentration

Answer 6

decreases protein solubility

Question 7

Uses the size difference between proteins and small molecules to separate them

Answer 7

dialysis

Question 8

Dialysis uses a

Answer 8

• semipermeable membrane
• dialysis bag submerged in buffer solution without small molecules = diffuse down concentration gradient
• protein molecules retained in the bag
• small molecules pass through the bag into the external solution

Question 9

Dialysis can

Answer 9

change one buffer for another

• permits changes to be made in the pH of the protein solution

Question 10

Uses differences in the sizes of proteins

Answer 10

gel-filtration chromatography

Question 11

Gel-filtration chromatography uses a column of

Answer 11

porous beads of highly hydrated polymer gel

Question 12

Examples of polymers in beads

Answer 12

• dextran (polycarbohydrate)
• agaraose (polycarbohydrate)
• polyacrylamide

Question 13

In gel-filtration chromatography, the proteins will flow down through the column and will be collected as

Answer 13

fractions

Question 14

The flow of … proteins is slowed by the beads, permeates pores of beads

Answer 14

smaller

Question 15

…proteins flow faster around the beads in the column

Answer 15

larger

Question 16

Summary of gel-filtration chromatography

Answer 16

• Biggest proteins first through the Beads
• large quantities of proteins can be separated
• separation not well resolved unless there’s a big difference between the sizes

Question 17

Ultimately uses differences in the masses of proteins

Answer 17

ultracentrifugation

Question 18

In ultracentrifugation, proteins are separated according to their

Answer 18

sedimentation coefficient

Question 19

Sedimentation coefficient

Answer 19

• proportional to protein mass

* heavier proteins sediments down faster

Question 20

Acts against centrifugal force

Answer 20

• buoyancy

* related to protein density

Question 21

Type of ultracentrifugation

Answer 21

zonal centrifugation
• AKA band centrifugation
• AKA gradient centrifugation

Question 22

Zonal centrifugation requires … inside the centrifuge tube

Answer 22

density gradient

• suppresses convection currents in the tube

Question 23

Can be produced by mixing low- and high-density solutions

Answer 23

density gradient

Question 24

Ultracentrifugation separates the proteins into

Answer 24

bands • collected as fractions • heaviest collected first

Question 25

It is possible to separate proteins by differences in their

Answer 25

surface electric charge

Question 26

pI – Isoelectric point

Answer 26

value of pH when the net surface charge is zero for that protein

Question 27

Proteins placed into the gel move within the applied electric field according to their

Answer 27

surface charge

Question 28

Something is isoelectrically focused when

Answer 28

the surrounding pH equals the pI vale for any given point, protein will cease to move in the field •high resolution degree of separation

Question 29

requires a pH gradient gel

Answer 29

isoelectric focusing | • uses a gel of polyampholytes

Question 30

Polyampholyte

Answer 30

small multi-charged polymers with different values of pI | • applying electric field creates pH gradient

Question 31

Low pH at

Answer 31

positive (+) anode

Question 32

high pH at

Answer 32

negative (-) cathode

Question 33

Proteins have differing numbers of

Answer 33

acidic and basic residues | • often have an overall positive or negative charge

Question 34

Uses beads with a surface charge chemically attached to them

Answer 34

ion-exchange chromatography | • beads typically cellulose or aragose

Question 35

Negative beads

Answer 35

Carboxymethyl – CM cellulose

Question 36

Positive beads

Answer 36

Diethylaminoethyl – DEAE cellulose

Question 37

Proteins with positive charge attach to

Answer 37

negatively charged beads | • other proteins pass down the column unhindered

Question 38

Positively charged proteins can hen be ... from the column

Answer 38

eluted | eluting = releasing

Question 39

Eluting bound proteins

Answer 39

* add low concentration of salt (sodium highly positive) * sodium ions bind to the beads instead of the proteins * weakly positive proteins elute off first * increase salt concentration = more positively charged proteins elute from column * all positively charged proteins can be collected as fractions as they come off the column

Question 40

Separates proteins according to size by applying an electric charge through a polymer gel

Answer 40

gel electrophoresis

Question 41

PAGE

Answer 41

Polyacrylamide Gel Electrophoresis • keeps from “falling down” • forms spaghetti-like strands

Question 42

Most common forms of gel electrophoresis uses

Answer 42

Sodium Dodecyl Sulphate – SDS | SDS-PAGE

Question 43

SDS is an

Answer 43

anionic detergent • disrupts all non-covalent interactions • binds to amino acid residues in 1SDS : 2amino acid • all proteins become negatively charged

Question 44

The negative charge on each protein becomes directly proportional to its

Answer 44

mass

Question 45

Beta-mercaptoethanol added to disrupt

Answer 45

disulfide bonds | • proteins become fully denatured

Question 46

Protein mixture flows down the gel...

Answer 46

from the cathode toward the anode • large proteins impeded in the gel by the strands • Smallest proteins fastest through the Strands

Question 47

In gel electrophoresis, the protein separation provides a direct measurement of their

Answer 47

masses | • proteins with known molecular masses run as a scale marker

Question 48

Makes use of the fact that many proteins tightly bind small specific molecules as part of their function

Answer 48

affinity chromatography

Question 49

...binds glucose very tightly

Answer 49

Concanavalin A

Question 50

Affinity chromatography overview

Answer 50

* covalently attach glucose to beads in a column * pass crude protein mixture w/ Concanavalin A down the column * Concanavalin A will bind to the glucose on the beads * all remaining proteins pass through unhindered Concanavalin A can be removed by passing ... down the column concentrated solution of glucose • binds better to “free” glucose than to the “bound” glucose on the beads •concanavalin A will elute from the column bound to the free glucose • the free glucose would be removed by dialysis

Question 51

Specific activity

Answer 51

Enzyme activity & protein concent | • the ratio of enzyme activity to the amount of protein in the mixture

Question 52

Forming homogenate with centrifuge

Answer 52

* dense pellet at bottom | * light supernatant at top

Question 53

Deferential centrifugation

Answer 53

Yields several fractions of decreasing density, each with hundreds of different proteins • to homogenate

Question 54

Eluting proteins in ion-exchange chromatography

Answer 54

* increase concentration of sodium chloride ( or other eluding buffer) * compete with positively charged groups on the protein for binding to the column * low density of net positive charge emerge first * followed by those with a higher charge density

Question 55

Cation exchange

Answer 55

(Ion exchange chromatography) | • positively charged groups bind to anionic beads

Question 56

Cationic proteins can be separated by chromatography on

Answer 56

Negatively charged carboxymethylcellulose | (CM-cellulose) columns

Question 57

Anionic proteins an be separated by anionic exchange on

Answer 57

Positively charged diethylaminoethylcellulose (DEAE-cellulose) columns