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Basic Biochemistry > BB5&6 Protein Isolation & Purification > Flashcards

BB5&6 Protein Isolation & Purification Flashcards

0
Q

The proteins in solution will denature unless they are stabilized against

A
  • changes in pH (add buffer)
  • changes in temperature (cool to 4c)
  • protease degradation (add protease inhibitors)
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1
Q

Protein Accessibility

A
  • in solution
  • in extra-cellular medium
  • intra-cellular = must be broken (lysis)
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2
Q

Protein separation uses differences in

A
  • solubility
  • size
  • charge
  • specific binding affinity
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3
Q

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

A

assay

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4
Q

Salting out

A
  • uses solubility differences
  • different proteins salt out at different salt concentrations
  • only partial separation
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5
Q

Solubility changes with addition of

A

ionic salts

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6
Q

Increasing salt concentration

A

decreases protein solubility

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7
Q

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

A

dialysis

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8
Q

Dialysis uses a

A
  • 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
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9
Q

Dialysis can

A

change one buffer for another

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

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10
Q

Uses differences in the sizes of proteins

A

gel-filtration chromatography

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11
Q

Gel-filtration chromatography uses a column of

A

porous beads of highly hydrated polymer gel

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12
Q

Examples of polymers in beads

A
  • dextran (polycarbohydrate)
  • agaraose (polycarbohydrate)
  • polyacrylamide
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13
Q

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

A

fractions

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14
Q

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

A

smaller

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15
Q

…proteins flow faster around the beads in the column

A

larger

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16
Q

Summary of gel-filtration chromatography

A
  • 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
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17
Q

Ultimately uses differences in the masses of proteins

A

ultracentrifugation

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18
Q

In ultracentrifugation, proteins are separated according to their

A

sedimentation coefficient

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19
Q

Sedimentation coefficient

A
  • proportional to protein mass

* heavier proteins sediments down faster

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20
Q

Acts against centrifugal force

A
  • buoyancy

* related to protein density

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21
Q

Type of ultracentrifugation

A

zonal centrifugation
• AKA band centrifugation
• AKA gradient centrifugation

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22
Q

Zonal centrifugation requires … inside the centrifuge tube

A

density gradient

• suppresses convection currents in the tube

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23
Q

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

A

density gradient

24
Q

Ultracentrifugation separates the proteins into

A

bands
• collected as fractions
• heaviest collected first

25
Q

It is possible to separate proteins by differences in their

A

surface electric charge

26
Q

pI – Isoelectric point

A

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

27
Q

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

A

surface charge

28
Q

Something is isoelectrically focused when

A

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

29
Q

requires a pH gradient gel

A

isoelectric focusing

• uses a gel of polyampholytes

30
Q

Polyampholyte

A

small multi-charged polymers with different values of pI

• applying electric field creates pH gradient

31
Q

Low pH at

A

positive (+) anode

32
Q

high pH at

A

negative (-) cathode

33
Q

Proteins have differing numbers of

A

acidic and basic residues

• often have an overall positive or negative charge

34
Q

Uses beads with a surface charge chemically attached to them

A

ion-exchange chromatography

• beads typically cellulose or aragose

35
Q

Negative beads

A

Carboxymethyl – CM cellulose

36
Q

Positive beads

A

Diethylaminoethyl – DEAE cellulose

37
Q

Proteins with positive charge attach to

A

negatively charged beads

• other proteins pass down the column unhindered

38
Q

Positively charged proteins can hen be … from the column

A

eluted

eluting = releasing

39
Q

Eluting bound proteins

A
  • 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
40
Q

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

A

gel electrophoresis

41
Q

PAGE

A

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

42
Q

Most common forms of gel electrophoresis uses

A

Sodium Dodecyl Sulphate – SDS

SDS-PAGE

43
Q

SDS is an

A

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

44
Q

The negative charge on each protein becomes directly proportional to its

A

mass

45
Q

Beta-mercaptoethanol added to disrupt

A

disulfide bonds

• proteins become fully denatured

46
Q

Protein mixture flows down the gel…

A

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

47
Q

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

A

masses

• proteins with known molecular masses run as a scale marker

48
Q

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

A

affinity chromatography

49
Q

…binds glucose very tightly

A

Concanavalin A

50
Q

Affinity chromatography overview

A
  • 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

51
Q

Specific activity

A

Enzyme activity & protein concent

• the ratio of enzyme activity to the amount of protein in the mixture

52
Q

Forming homogenate with centrifuge

A
  • dense pellet at bottom

* light supernatant at top

53
Q

Deferential centrifugation

A

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

54
Q

Eluting proteins in ion-exchange chromatography

A
  • 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
55
Q

Cation exchange

A

(Ion exchange chromatography)

• positively charged groups bind to anionic beads

56
Q

Cationic proteins can be separated by chromatography on

A

Negatively charged carboxymethylcellulose

(CM-cellulose) columns

57
Q

Anionic proteins an be separated by anionic exchange on

A

Positively charged diethylaminoethylcellulose (DEAE-cellulose) columns

Basic Biochemistry (15 decks)

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