Chapter 5 - Lab Techniques

Question 1

Proteomics

Answer 1

study of the cellular protein complement at varying times or conditions

a. Leads to the identification of new proteins
b. Human genome sequencing allows identification of potential proteins through computer algorithms and similarity searches

Question 2

High frequency amino acids

Answer 2

ALV

8.3/9.7/6.9

Question 3

Low frequency amino acids

Answer 3

WHC

1.1/2.3/1.4

Question 4

Size range of proteins

Answer 4

3,427 (Proteinase Inhibitor III (bitter gourd))

3,816,188 (Titin (human))

Protein size and range is limited by the requirments for adopting a 3D structure

Question 5

Protein molecules: study of structure and function requires isolation and purification for characterization

Answer 5

1. size too large for organic synthetic methods - must be produced by a living organism
2. most analytical techniques and assays of activity require about 95-98% purity
3. Vulunerable to damage by many factors: environmental (pH, temp, surface exposure), to enzymatic degradation by proteases
4. Possible limited availability of target protein for study

(requires harves from tissure or organism or origin and target protein expression by recombinant methods (in a foreign organism))

5. Sepatartion techniques fro protein purification exploit protein properties due to amino acid sequence and composition

Question 6

Fluorescent tag

Answer 6

Add a fluorescent tag to your target protein via genetic engineering or recombinant DNA cloning techniques (plasmid vectors with green fluorescent or other fluorescent protein gene sequences included)

Detection method

Question 7

UV spectroscopy

Answer 7

detects aromatic amino acids within a protein sequence

1) Beer’s law to determin concentration if molar extinction coefficient is known (possible downfall)
2. Detection limits: as low as 50-100 ug protein/mL
3. Does not destroy proteins in the solution (good)

Detection method

Question 8

Dye-binding to protein surfaces or peptide bonds

Answer 8

Two common assays:

1. Coomassie G-250 (Bradford Assay): binds to positiviely charged amino acids on the protein surface
2. Bichichinonic acid: Reacts with peptide bonds
   a. Light absorption max for protein bound dye shifts relative to unbound dye
   b. detects protein down to 1 ug/mL (good)
   c. Destroys the protein in the sample (bad)

Detection method

Question 9

ELISA

Enzyme-Linked Immunosorbent Assay

Answer 9

immunohistochemical technique

1. Antibodies specific to target protein’s 3D shape, physical and chemical properties are immobilized on a solid surface
2. Protien mixture containing target protein incubated on anitbody-bound surface
3. Unbound proteins and molecules washed away
4. Bound proteins are detected by addition of a 2nd antibody specific to either the target protein or available primary antibody or affinity tag to promote detection (many variations available)

HIV ELISA

Detection method

Question 10

Protein Purification Strategy

Answer 10

series of steps using a combination of techniques designed to separate a target protein out of a protein mixture (cell or tissue) -> yield: pure protein “reagent”

All purification methods take advantage of the varialbe physical and chemical properties arising from their unique amino acid dquence, composition, and shape

Some common fractionation methods designed to isolate a protein from other contaminants exploit differences in solubility, ionic charge, polarity, size, or binding specificity

Question 11

Protein Characteristic

solubility

ionic charge

polarity

size

binding specificity

Answer 11

Purification procedure

salting out

ion exchange chromatography; electrophoresis; isoelectric focusing

hydrophobic interaction chromatography

gel filtration chromatography; SDS-PAGE

affinity chromatography

Question 12

“Salting-Out”

Ammonium sulfate precipitation

Answer 12

1. Proteins with hydrophilic surface require solvation by water to remain dissolved in aqueous solutions
2. High salt concentrations in the solution compete for water solvation
3. Unsolvated proteins aggregate and form precipitates based on their solubiilty at a given salt concentration
4. Precipitated proteins are collected by centrifugation: either the precipitate or the supernatant may contain the target protein
5. Proteins are least soluble at their isoelectric point, a characteristic that enhances the technique’s effectiveness for particular proteins

NH4SO4 - hydrophboic inside peaks out

Question 13

Column Chromatography

Answer 13

exploits the molecue partitioning between a stationary and mobile phase

1. Ion exchange
2. Gel filtration
3. Affinity chromatography

Question 14

Ion-Exchange Chromatography

Answer 14

separation based on ionic charge

1. DEAE (positive) - anion-exchange (Q-matrices strong)(quaternary amines)
2. CM (negative) - cation-exchange (weak) (S-matrices strong)(sulfate groups)

Proteins bind charged column matrix at low ionic strength solutions and are eluted at high ionic strength. Buffer pH can also alter protein charges and column affinity.

Question 15

Gel Filtration Chromatography

Answer 15

aka size-exclusion chromatography

separates molecules based on size differences (and/or radius) with a porous matrix (pores range in size)

a) Pores exclude larger molecules that cannot fit but include smaller molecules
- Large proteins elute first from a sample mixture after a shorter path through matrix
- Smaller included molecules elute based on their ability to fit into the matrix pores, gving them a longer path through the beads.
b) The rate of protein elution is based on the extent to which a protein is included or exclued in a gel matrix with a characterized range of pore sizes (cross-linked gel polymers)

Can be used to tell you something about your protein’s size

Question 16

Affinity Chromatography

Answer 16

Exploits specific non-covalent binding behavior to a specific ligand immobilized on the solid support

a) immobilized ligands: substrate analogs, antibodies specific to the protein or protein’s affinity tag, or a metal ion specific to a protein characteristic
b) Highly specific separation efficiency; expensive reagents
c) Cooperative technique now widely used: recombinant proetin genectically engineered or recombinant proteins to express an “affinity-tag” specific to a complimentary resin

His 6 or 9 added to a terminus - specifically binds to immboilzed Ni2+

FLAG tag on protein specific to the FLAG-specific antibody

Proteins are eluted with buffers containing competitor molecules for resin binding sites

Question 17

Native Polyacrylamide Gel Electrophoresis (PAGE)

Answer 17

1) Separates molecules based on size and charge in an electric field
2) protein retain 3D structure

Question 18

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Answer 18

1) Molecule separation based on mass
- proteins are denatured by heating and linearized in the presence of the negatively-charged SDS detergent -> most proteins bind a similar ration of SDS to protein (equal distribution) (unfolds protein and S- stabilizes it)
b) SDS treated samples are loaded on a porous gel matrix and electrical current is applied
c) Negatively charged protein molecules migrate through the gel at a rate proportional to the log of their molecular mass
d) Protein molecules are visualized in the gel: Coomassie blue dye stain or silver straining

Question 19

2D Gel Elctrophoresis

Answer 19

Combines SDS-PAGE with a second type of electrophoresis such as isoelectric focusing: proetins migrate through a gel matrix with a pH gradient

1) SDS PAGE gel separates proteins by size
2) Gel turned 90 degress in orientation or current applied from a different direction to run proeins through the gel matrix under different conditions such as a pH gradient
3) Unknown protein “spots” can be exised and identified by methods such as: Mass spec; protein sequencing
4) Protein sequence comparison to protein sequence databases using multiple sequence alignments and sequence similarity searches