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Biochemistry > Protein Purification And Characterization Techniques > Flashcards

Protein Purification And Characterization Techniques Flashcards

1
Q

Give the 3 Techniques for Purification of Protein.

A
  1. Protein Extraction from cells
  2. Column Chromatography
  3. Electrophoresis
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2
Q

3 Types of Protein Extraction from the cell

A
  1. Homogenization
  2. Differential centrifugation
  3. Salting-out
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3
Q

Homogenization involves _____ and _____ the cell.

A

Breaking and opening

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4
Q

Homogenization involves breaking and opening the cell that can be done several ways: What are the 4 ways?

A

Grinding the tissue in a blender with suitable buffer
Using potter-elvehjem homogenizer
Sonicating (apply sound energy to agitate the cell)
Cycles of freezing and thawing

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5
Q

What is Centrifugation?

A

It is a technique wherein it use for the separation of particles from a solution or the sample according to their size, shape, density, viscosity.

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6
Q

It involves spinning the sample in centrifuge at different speed.

A

Differential Centrifugation

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7
Q

BONUS: About Differential Centrifugation

A

Differential centrifugation depends on what protein interest is, the precipitate or supernatant liquid may be discarded.

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8
Q

Imagine the process of centrifugation:
1.0 In a test tube there is a ____ with a buffer.
Using a rod it rotates clockwise and up/down. This process help to _____ the cell.

A

Sample.

Destroy.

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9
Q

Imagine the process of centrifugation:
1.1 After destroy the cell, the sample will filter and put to another test tube. What is the liquid in the test tube after filtration happens?

A

It is the residues (with a buffer)

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10
Q

Imagine the process of centrifugation:
1.2 The test tube put in the centrifuge about 10min. The test tube now having a two difference: the liquid and the soft solid at the bottom.

What is the liquid and the solid formed after centrifugation?

A

Liquid is the supernatant (which is the protein)

In the bottom is the residues (nuclei unbroken cells)

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11
Q

Imagine the process of centrifugation:

1.3 Example you gather your desire in supernatant 4. What will happen to the residues in supernatant 1,2, and 3?

A

It will be discarded.

Note: It depends on what you looking for it dictates where do you stop.

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12
Q

Salting-out.

It is a process of lowering the solubility of protein. What salt will you use to lower the solubility?

A

(NH4)2 SO4 or ammonium sulfate

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13
Q

Addition of (NH4)2 SO4 will decrease the solubility of the protein by taking-away most of ____ molecules through ion-dipole interaction.

A

H2O or Water

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14
Q

The amount of H2O molecules will dehydrating the protein causing the molecules to have _____ interaction with each other.

a. Hydrophilic
b. Hydrophobic

A

b. Hydrophobic

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15
Q

It is a technique use to separate components of mixture?

A

Column chromatography

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16
Q

Chromatography comes to the Greek word chroma means ____ and graphein means _____

A

color; to write

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17
Q

2 Phases found in the column

A
  1. Mobile Phase

2. Stationary phase

18
Q

Mobile Phase is also known as _____

A

Eluent

19
Q

3 Types of Column Chromatography in separation of Protein

A
  1. Size-exclusion Chromatography
  2. Affinity Chromatography
  3. Ion-exchange chromatography
20
Q

It is known as gel-filtration chromatography.

It uses gel particles in a beads form packed in a column.

A

Size-exclusion Chromatography

21
Q

What is the basis of separation of Size-exclusion chromatography?

A

Size of the molecule

Note: Much bigger molecule will be eluted first (will go out first in the column).

22
Q

BONUS: This is a process in Size-exclusion Chromatography.

A

Imagine: In a column, there’s a bead, small and large molecules. When the column run the large molecule will travel the beads until it passed thru first in the column. Then, small molecules will next…

23
Q

It uses polymeric material as stationary phase.

Ligand binds specifically to desired protein.

A

Affinity Chromatography

24
Q

What is the basis of separation of Affinity Chromatography?

A

Affinity of the protein to the ligand

25
Q

In Affinity Chromatography. Proteins with affinity to ligand will _____?

a. eluted first
b. not eluted first

A

b. not eluted first

Protein with OUT affinity will eluted first.

26
Q

In Affinity chromatography. Bound protein is eluted by?

A

Flush high concentrated eluent to push the protein to the column.

Changing the pH or ionic strength of the mobile phase.

27
Q

BONUS: This is a process in Affinity Chromatography.

A

Imagine: There is a substance (S) at the bottom of the column and the sample mixed proteins in the top.
eg. X1, X2, X3.mwhich is the sample proteins
Now, when column run X2 and X3 will be eluted first but X1 will bind to the “S”. To elute the X1 we need to put a high concentration to the eluent. That X1 is the desired protein by affinity.

28
Q

What is the basis of separation of Ion-exchange Chromatography?

A

Net charge of the protein.

29
Q

It is a negatively charged ion-exchange resin.

a. Cation exchanger
b. Anion exchanger

A

a. Cation exchanger

30
Q

It is a positively charged ion-exchange resin.

a. Anion exchanger
b. Cation exchanger

A

a. Anion exchanger

31
Q

3 Common resins in Cationic-exchange

A
  1. Polystyrene resin (Dowex-50) :strongly acid
  2. Carboxymethyl (CM) Cellulose :weakly acid
  3. Polystyrene resin ;weakly acid chelating
32
Q

2 Common resins in Anionic-exchange

A
  1. Polystyren resin (Dowex-1) :strongly base

2. Diethylamioethyl (DEAE) cellulose :weakly base

33
Q

Ions that binds to cation-exchange resin?

A

Na+ and K+ ions

34
Q

Ion that binds to anion-exchange resin?

A

Cl- ion

35
Q

Protein with no net charge or have a same charge with the resin will _____.

a. last be eluted
b. eluted first

A

b. eluted first

36
Q

It is based on the migration of the charge particles in an electric field toward an electrode of opposite charge.

A

Electrophoresis

37
Q

Medium use in Electrophoresis?

A

Agarose or polyacrylamide gels

38
Q

Method use to estimate the purity and molecular weight of the protein sample.

One of the type of electrophoresis.

A

SDS-PAGE

39
Q

Basis of separation of SDS-PAGE?

A

Mass or molecular weight of protein.

The smaller protein move faster. Opposite of size-extraction

40
Q

This method takes advantage of different isoelectric points of protein.

A

Isoelectric Focusing

41
Q

What is the basis of separation in Isoelectric focusing?

A

pI of proteins

42
Q

BONUS: This is a process in Isoelectric Focusing.

A

Imagine: Gel is prepared with pH gradient corresponding to the electric field. Protein migrates through the gel under the influence of electric field until it reach the pH equivalent to the pI.

Biochemistry (12 decks)

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