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Biochemistry > Class 7 E1 > Flashcards

Class 7 E1 Flashcards

1
Q

Why use protein purification?

A

research, use in drugs, additives in detergents, foods, industrial processes.

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2
Q

As purification proceeds then?

A

of components / contaminants decreases.

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3
Q

Protein purification depends on?

A

size / charge.
location in cell (nucleus, membrane, mitochondrion)
solubility (hydrophobic / philic).

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4
Q

When do you test the purity of the same?

A

at the end.

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5
Q

Steps of protein purification?

A
  1. homogenize cells.
  2. crude separation.
    3, 4, 5. refined separation + purification steps.
  3. access success.
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6
Q

protein purification: fold purification

A

amount of protein of interest in final sample / amount in crude sample.

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7
Q

protein purification: specific activity

A

measure of enzyme purity, units catalyzed / time / mg enzyme.

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8
Q

protein purification: homogenization (sonification)

A

grinding / disrupting tissue to make uniform sample.

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9
Q

protein purification: centrifugation

A

spinning tube fast enough so heavier particles separate from lighter particles.

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10
Q

protein purification: yield (%)

A

total amount of protein (or activity) in final sample / amount of protein (or activity) in crude sample.

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11
Q

salting in / out

A

basic (not refined) way to separate class A from B.

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12
Q

Salt amount in salting in / out?

A

little - helps keep charged molecules in soln.

a lot - makes charged molecules precipitate.

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13
Q

What are the 3 methods used to purify proteins?

A
  1. Salting out
  2. Column-based methods
  3. Immunoprecipitation
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14
Q

What are the 3 types of column-based methods?

A
  1. size-exclusion chromatography
  2. ion-exchange chromatography
  3. affinity chromatography
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15
Q

chromatography

A

movement of a compound w/ mobile phase and stationary phase.

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16
Q

chromatography: mobile phase

A

medium, compound dissolved and applied to stationary phase (buffer).

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17
Q

chromatography: stationary phase

A

immobile / matrix over which mobile phase containing compound flows (“matrix””

18
Q

Separation occurs in chromatography bc?

A

diff compounds have diff relative affinities for mobile vs. stationary phases.

19
Q

chromatography columns

A

allow separation of protein soln into fractions.

20
Q

chromatographic media

A

resins / beads, microscopic - form support / separation chemistry, size of beads (mesh size) = diameter of any particle - #’s reciprocally related.

21
Q

What are placed in chromatography columns?

A

resins

22
Q

size exclusion chromatography

A

separates proteins based on size

23
Q

The gel beads in size exclusion chromatography have pores of a defined size range that?

A

allow smaller molecules to enter while excluding larger molecules.

24
Q

Which proteins reside longer in the gel beads in size exclusion chromatography?

A

smaller ones.

25
Q

Which proteins elute in early fractions of size exclusion chromatography?

A

large ones.

26
Q

ion exchange chromatograpahy

A

separates based on charge

27
Q

the stationary phase of ion exchange chromatography is?

A

charged and binds proteins of opposing charge.

28
Q

In ion exchange chromatography you collect?

A

fractions of eluate from column over time.

29
Q

In ion exchange chromatography you can elute using what?

A

salt, pH change.

30
Q

What are the steps of ion exchange chromatography?

A
  1. add mix of AA’s or proteins into an immobilized cationic (+) / anionic (-) stationary phase.
  2. the opp charged proteins will bind to the charged matrix as they flow through.
  3. Elute using a specific buffer.
31
Q

affinity chromatography

A

isolates specific proteins and purifies fusion proteins.

32
Q

Affinity chromatography takes advantage of?

A

specific interactions btw protein and stationary phase.

33
Q

The stationary phase in affinity chromatography can be loaded with?

A

a ligand or antibody; natural ligand of protein / antibody.

34
Q

Some of the epitope tags that can be engineered to express the vector or transgene in affinity chromatography.

A
  1. 6x-His
  2. GST
  3. MBP
  4. Protein A
    etc.
35
Q

Steps of using affinity chromatography to purify fusion proteins.

A
  1. amylose resin matrix put into column.
  2. run homogenate over column.
  3. wash of other proteins.
  4. collect fractions
36
Q

affinity chromatography: Factor X

A

can cleave and release pure target protein.

37
Q

immunoprecipitation

A

uses antibodies

38
Q

Immunoprecipitation uses?

A

Ig Fc linked to beads

39
Q

The Ig Fc linked beads in immunoprecipitation work like?

A

the stationary phase in a column similar to how Ig’s are embedded in the B cell membrane.

40
Q

The Ig Fc linked beads are heavy bc?

A

they allow quick separation via quick centrifugation spin (<1 min)

41
Q

Steps to immunoprecipitation?

A
  1. prepare sample.
  2. capture antibody.
  3. wash and elute.

Biochemistry (10 decks)

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