LAB(4) - PROTEIN ISOLATION TECHNIQUES

Question 1

Protein purification

Answer 1

a series of processes intended to isolate one or few protein form a complex mixture, usually cells, tissues or whole organisms

Question 2

an essential 1st step in understanding their function

proteins must be released from the cell to be purified

based on the basic properties of protein like solubility (salt, pH, temperature), size, charge, and binding properties (ligands)

Answer 2

protein purification

Question 3

What are the general steps in protein purification?

Answer 3

• selection of a protein source
• tissues homogenization and solubilization
• stabilization of proteins
• isolation techniques utilize different properties of protein

Question 4

Homogenization

Answer 4

the process requires the disruption of the outer cell membrane,
intracellular membranes and the surrounding
extracellular structures.

Question 5

(huh?) For proteins from muscle that would mean grinding it up, for an intercellular protein that would mean breaking the cells open, etc. This is
always done in the presence of a buffer and inhibitors.

Question 6

What does the use of detergent solution do?

Answer 6

increase the
solubility of proteins (for protein membrane
extraction). Help stabilize and solubilize
proteins as they are released from the cell.

Question 7

What does the use of other reagents (chaotropic agents) such as urea and guanidine HCl (they breakdown the structure of the protein and dissolve well in water) do?

Answer 7

Help break apart the lipid cell membrane

Question 8

Salting in

Answer 8

refers to the increase of proteins
solubility in a solution with low salts concentration.

Question 9

Low salts concentration, solubility of protein increases.

Question 10

Salt molecules stabilize protein molecules by?

Answer 10

Decreasing the electrostatic energy between the
protein molecules which increase the solubility of
proteins. (salting in)

Question 11

Salting out

Answer 11

the precipitation of the
proteins at high salts concentration. It is a
purification method that relies on the basis of protein solubility (reducing the solubility)

Question 12

High salt concentrations

• increase ionic strength of a protein solution
• decrease protein solubility thus precipitation

Question 13

a purification method at initial molecule purification that lacks the ability for precise
isolation of a specific protein.

Answer 13

Salting out

Question 14

Ammonium sulfate

Answer 14

commonly used salt

Question 15

Why ammonium sulfate?

Answer 15

1. large solubility in water
2. relative freedom from temperature effects
3. no harmful effects on most of the proteins

Question 16

The amount of salt needed to isolate a specific protein is determined from the salt’s fractionation table

Question 17

study the Hofmesiter series !!!

Question 18

the proteins are separated after salt addition by?

Answer 18

Centrifugation

Question 19

Centrifuging the liquid containing the cells in a high-density medium may precipitate the desired cells depending on the density of each constituent cell.

Question 20

additionally
applicable for eliminating undesired cellular impurities or obtaining certain cell organelles.

Answer 20

Density-gradient ultracentrifugation

Question 21

Dialysis

Answer 21

Removal of salt
molecules from the
isolated protein solution through a semi
permeable dialysis bag

Question 22

The salt molecules move from the more
concentrated solution
(from inside the dialysis bag) to the less
concentrated solution.

Question 23

Buffers and salts exchange until an equil. is established b/w the inside and outside if the membrane; this process could not distinguish b/w proteins effectively.

Question 24

Chromatography

Answer 24

a separation technique used to separate complex mixtures of amino acids.

Question 25

Proteins are separated using column chromatography
(..in particular high-performance liquid
chromatography (HPLC)) because?

Answer 25

proteins tend to
denature when separated using TLC.

Question 26

What are the advantages of HPLC over TLC?

Answer 26

1. Resolution and sensitivity
2. Quantitative analysis
3. Selectivity and versatility
4. Automation and reproducibility
5. Speed and throughput

Question 27

Chromatography

Answer 27

based on the principle where molecules in mixture applied onto the surface or into
the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid
of a mobile phase

Question 28

one of the most powerful fractionation methods.

Answer 28

Column chromatography

Question 29

Column chromatography can separate components o mixtures based upon?

Answer 29

• adsorption (liquid-solid)
• partition (liquid-solid)
• affinity
• differences among their molecular weights

Question 30

Gel filtration chrom. (GFC)

Answer 30

a form of partition
chromatography used to separate molecules of different molecular size.

Question 31

GFC:

• small molecules = freely enter internal solvent space of the gel bead
• larger molecules = too large to penetrate the gel pores and travel b/w beads and elute first

Question 32

Ion exchange (by charge)

Answer 32

Proteins may have net positive or negative charge at different
pH range. If a protein has a net positive charge, negatively
charged molecules bind to positively charged solid supports
and positively charged molecules bind to negatively charged

Question 33

Anion exchange resins (positive charge)

Answer 33

separate negatively charged compounds

Question 34

Cation exchange resins (negative charge)

Answer 34

separate positively charged compounds

Question 35

Affinity chromatography (by specific binding affinity)

Answer 35

a separation method based on a specific binding
interaction between an immobilized ligand and its binding partner.

Example enzyme/substrate,
receptor/ligand, enzyme/inhibitor,
antibody/antigen interactions.

Question 36

Affinity chromatography (by specific binding affinity)

Answer 36

High selectivity, resolution, and capacity in protein
purification schemes can be easily achieved with affinity chromatography.

Question 37

How does affinity chrom isolate targeted proteins?

Answer 37

1. attaching the chemical grp. which could affinity with target protein to the column
2. Loading the sample into the column and let the target protein affinity with chemical grp.
3. eluting the target protein by high concentration

Question 38

Affinity chrom.:

1. the ligand is immobilized onto a solid support matrix
2. the crude extract is passed through the column
3. the target molecule for which the ligand possess affinity is retained
4. all other material is eluted
5. the bound target protein is eluted by alteration of the mobile-phase conditions

Question 39

High performance liquid chromatography

Answer 39

a process of separating components in a liquid
mixture. A liquid sample is injected into a stream of solvent (mobile phase) flowing through a column packed with a separation medium
(stationary phase).

Question 40

High performance liquid chromatography

Answer 40

• The flow rate of the solvent is set through
  computer input and controlled by pumps.
• The peak represent different kinds of compounds and result is high resolution and rapid separation

Question 41

study on the HPLC flow diagram, overview,

Question 42

HPLC:

Once in the column, the sample mixture separates as a result of different components adhering to or diffusing into the gel in form of different zones called “bands”

Question 43

Mass spectrometry

Answer 43

used to identify
unknown compounds via molecular weight
determination, to quantify known compounds,
and to determine structure and chemical
properties of molecules.

Question 44

Edman degradation

Answer 44

classic method to
sequentially removes one amino acid at a time from the N-terminus of a protein, which is then
identified.

Question 45

Electrophoresis

Answer 45

a separation technique used to separate proteins based on charge and size by applying an electric field

Question 46

Protein electrophoresis

Answer 46

typically carried out in a gel matrix

Question 47

Polyacrylamide gel electrophoresis (PAGE)

Answer 47

carried out in a gel which serves as a molecular sieve

Question 48

study the two forms of electrophoresis

Question 49

SDS-PAGE

Answer 49

• protein mixture is heated in the presence of 2-mercaptoethanol and SDS
• unfolded polypeptide chains will then migrate towards the anode
• smaller polypeptides migrate further through the gel than larger one

Question 50

Proteins are purified from a mixture using combination of several techniques based on protein properties.

protein property = technique

Answer 50

solubility = differential precipitation

molecular size = gel filtration chromatography

molecular charge = ion exchange chromatography

hydrophobicity = reversed phase HPLC

biological activity = affinity chromatography

Question 51

Trivia:

Answer 51

• The first breakthrough in speeding up protein synthesis
  was the development of a device in 1960 by R. Bruce
  Merrifield that could automatically synthesize
  polypeptide chains.
• Pancreatic ribonuclease (124 amino acid units) and
  human growth hormone (188 amino acid units) have
  been prepared with this technique
• Biological systems ( i.e. via genetic engineering) are
  currently being used and developed to speed up the
  process of protein synthesis.