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Biochemistry > Isolation/Purification of Proteins > Flashcards

Isolation/Purification of Proteins Flashcards

1
Q

What does Isolation/Purification of Proteins involves?

A

Disrupt the cell/cell membranes to obtain a cellular homogenate
(a “soup” containing proteins, membranes, and nucleic acids)

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2
Q

What are five different methods for isolation and purification of proteins?

A
  1. Mortar and Pestle
  2. Chemical
  3. Repeated Freeze/Thaw Cycles
  4. Ultrasonication
  5. High Pressure
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3
Q

What is centrifugation?

A
  1. is sedimentation of particles under the influence of the centrifugal force and it is used for separation of superfine suspensions.
  2. Starts off as homogenate form and forms a supernatant liquid.
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4
Q

What are two types of centrifuge?

A
  1. Differential (simple) centrifugation

2. Gradient Ultracentrifugation

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5
Q

What is Differential (simple) Centrifugation?

A

separation of items (cell organelles) based upon density.

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6
Q

Differential (simple) Centrifugation: centrifuge at 500 x g for 10 mins

A

pellet: nuclear fraction

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7
Q

Differential (simple) Centrifugation: centrifuge at 10,000 g for 20 mins

A

Pellet: mitochondrial fraction

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8
Q

Differential (simple) Centrifugation: centrifuge at 100,000 g for 1 hour

A

cytoplasm (soluble proteins) —> pellet: microsomal fraction

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9
Q

What is Gradient Ultracentrifugation?

A
  1. separation of proteins based upon
    sedimentation coefficient, s (density, mass, and shape).
  2. The
    medium commonly used is cesium chloride (CsCl).
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10
Q

Salting in

A
  1. At low concentrations, the presence of salt stabilizes the various
    charged groups on a protein molecule
  2. This enhances the solubility of
    protein.
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11
Q

Salting Out (Protein precipitation in the presence of excess salt)

A
  1. As the salt concentration is increased, a point of maximum protein
    solubility is usually reached.
  2. Further Increase in salt concentration = less water to solubilize protein
  3. Protein therefore precipitates if there is not enough water molecules to interact with protein
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12
Q

Different proteins precipitate “out” at different, ___________.

A

High Salt concentrations

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13
Q

What chemical is often used in salting out proteins?

A

(NH4)2SO4, ammonium sulfate

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14
Q

How can proteins be separated from molecules? (salting out)

A
  1. dialysis (w/ dialysis bag, concentrated solution, and buffer)
  2. Reaches equilibrium
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15
Q

What is the main fo

A

size exclusion

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16
Q

What is involved in Gel Filtration chromatography?

A
  1. Traps smaller molecules in the pores (bead) of a particle
  2. Larger molecules are too large to enter/pass through pores.
  3. Thus, larger molecules flow through column quicker
  4. Smaller molecule, longer the retention time
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17
Q

In ion-Exchange chromatography, what is retention based on?

A

attraction b/t charged sites bound to stationary phase and oppositely charged molecules

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18
Q

What gets excluded in ion-exchanged chromatography?

A

Molecules of the same charge get excluded (flow through)

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19
Q

In ion-exchanged chromatography, what do the ion exchangers favor?

A

Higher charged smaller molecules

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20
Q

In ion-exchanged chromatography, what is used to alter retention times and remove bound molecules?

A

counter ions and changes in pH

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21
Q

In ion-exchanged chromatography, what is cation exchange?

A

positively charged
molecules on a negatively-charged
stationary phase.

22
Q

In ion-exchanged chromatography, what is anion exchange?

A

negatively charged
molecules on a positively-charged
stationary phase.

23
Q

What is affinity chromatography?

A

A method of separating molecules based on a highly specific

biological interaction

24
Q

What are the biological interaction that can exist in affinity chromatography?

A
  1. antigen/antibody
  2. enzyme/substrate
  3. receptor/ligand
25
Q

What does IMAC stand for?

A

Immobilized Metal ion Affinity Chromatography

26
Q

How does Immobilized Metal ion Affinity Chromatography work?

A

by allowing proteins with an affinity for metal ions to

be retained in a column containing immobilized metal ions

27
Q

In IMAC, what happens when proteins are tagged?

A

attain metal binding characteristics

28
Q

In IMAC, how can proteins of interest be removed?

A
  1. changing pH

2. adding competitive molecule

29
Q

What is High Performance (pressure) Liquid chromatography?

A
  1. Highly improved formed of column chromatography
  2. Solvent is forced through under high pressure of up to 400 atmospheres (instead of being dripped under normal gravity)
30
Q

Why are smalled particle sizes used for column packing in high performance liquid chromatography?

A

Increases surface area for interactions b/t stationary phase and flowing molecules

31
Q

What are two types of phases that exist in high performance liquid chromatography?

A
  1. Normal Phase

2. Reverse Phase

32
Q

What is the normal phase?

A
  1. When the stationary phase is polar and solvent is non-polar.
  2. Retains polar molecules
33
Q

What is the reverse phase?

A
  1. When the stationary phase is non-polar and solvent is polar.
  2. Retains non-polar molecules
34
Q

Benefits of high performance liquid chromatography?

A
  1. Faster
  2. Better Separation
  3. High Resolution
35
Q

What is the purpose of electrophoresis?

A
  • Determine how pure protein preparation is

- Separation of proteins via a current/electrical charge

36
Q

What are three methods for electrophoresis?

A
  1. SDS-PAGE
  2. Native
  3. 2-D Gel Electrophoresis
37
Q

What is involved in SDS-PAGE?

A
  1. “Denatured Gel”

2. Gel contains SDS

38
Q

What is SDS?

A

12 carbon detergent w/ sulfur

39
Q

What does SDS do?

A
  1. Gives proteins net negative charge (b/c of negatively charged sulfate group)
  2. Denatures proteins (disrupts prevents H-bonding)
40
Q

What is PAGE and what is its purpose?

A
  1. consists of acrylamide and methylenebisacrylamide (2 versions of acrylamide)
  2. Persulfate is added for polymerization (cross links acrylamide)
  3. cross links create pores that proteins can move through
41
Q

What is the purpose of the pores created in PAGE?

A
  1. Allows proteins to pass through

2. Smaller proteins pass through faster than larger proteins (separation of proteins based on size)

42
Q

What causes the proteins to move in SDS-PAGE?

A
  1. Negatively charged pole at the top and positively charged pole at the bottom
  2. B/c of net negative charge of proteins, they move towards the positive pole
43
Q

What is the purpose of B(2)-mercaptoethanol and Dithothreitol in SDS-PAGE?

A
  1. Reducing agents

2. Reduces disulfide bridges to sulfhydryl groups between polypeptides

44
Q

What is the purpose of native electrophoresis combined with 2D gel electrophoresis?

A

Separate proteins based on:

  1. IP
  2. Shape
  3. Size
45
Q

How does native electrophoresis work?

A
  1. Separates proteins based on IP
  2. Done with PH gradient when proteins are still in their native form with native charges
  3. Proteins are loaded in wells (located in the middle of the structure)
  4. proteins move to the pole with opposite charge of the native charge (anode/cathode)
46
Q

What happens when a protein reaches its IP point in native electrophoresis?

A
  1. Its stops moving
  2. Net charge becomes 0 and neither pole affects movement of the protein
  3. pH becomes neutral
47
Q

What does native electrophoresis lack?

A

SDS and B-ME (native charges and disulfide bridges remain in proteins)

48
Q

What occurs in 2D electrophoresis?

A
  1. Separate proteins by pI value (native electrophoresis)
  2. Soak gel in SDS solution and fit it in “well” of SDS PA gel
  3. Turn on “power”
  4. Proteins separate by size/molecular weight (like in regular SDS PAGE electrophoresis)
49
Q

What is the end product in 2D gel electrophoresis?

A

Dots on gel representing a single protein (very pure proteins)

50
Q

What is the best way to determine how pure your protein preparation is?

A

Specific activity, if protein is an enzyme.

51
Q

What is the least efficient way of getting a purified protein?

A

Homogenization (large yield, less purified)

52
Q

What is the most efficient way of getting a purified protein?

A

Affinity chromatography (less yield, very pure sample)

Biochemistry (15 decks)

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