Tools of Biochemistry

Question 1

Proteomics

Answer 1

The study of proteins produced by the genome

Question 2

Differences between proteins and genome

Answer 2

1. Proteins are dynamic, whereas genome is fixed
2. # and type of proteins change in each cell, whereas genome is fixed
3. Proteins can change within cell under some conditions, whereas genome can’t change
4. Proteins interact, whereas genome is non-interactive

Question 3

Ways to study a protein

Answer 3

1. Sequence it
2. Run an assay that measures protein function
3. Quantitate protein (specific activity= enzyme activity/total protein)
4. Isolate protein from cells or tissue
5. Solve its structure
6. Identify its temporal and spatial environment

Question 4

Using color to analyze proteins

Answer 4

Product has color; reactant does not

Question 5

Differential centrifugation

Answer 5

Centrifuge at progressively higher speeds to separate out heavier materials of cell from lighter ones
Can be used to isolate spatially separated proteins

Question 6

Salt precipitation

Answer 6

Salt draws water away from protein

Without water to solubilize, protein becomes insoluble

Question 7

Dialysis

Answer 7

Semi-permeable membrane retains large molecules but lets small molecules into solution

Question 8

Gel filtration chromatography

Answer 8

Column is loaded with beads: small molecules enter the spaces within the beads, so they come off the column last (large molecules can’t enter the beads, so they just flow through)
Doesn’t actually capture anything; just slows things down

Question 9

Ion affinity chromatography

Answer 9

Either use cation exchanger (negatively charged) or anion exchanger (positively charged) beads
Oppositely charged proteins bind to bead and identically charged proteins flow through
Oppositely charged proteins are eluted with increasing concentrations of NaCl

Question 10

Affinity chromatography

Answer 10

Proteins attach to beads that contain residues of what they normally bind to
To release protein, add the actual substance that the protein binds to or add denaturants

Question 11

Electrophoresis

Answer 11

Proteins migrate on charged gel based on their size

Large friction due to large size or shape: less velocity and less movement

Question 12

Ingredients for gel electrophoresis

Answer 12

1. Polyacrylamide gel matrix
2. Buffer to hold uniform negative charge
3. Denaturing agents (beta mercaptoethanol or heat) to disrupt cysteine bonds
4. Anionic detergent (sodium dodecyl sulfate) to give proteins negative charge

Question 13

2D gel electrophoresis

Answer 13

Proteins are separated according to size and isoelectric point
Good separation of complex protein mixtures

Question 14

Isoelectric focusing

Answer 14

Principle of 2D gel electrophoresis
Proteins are separated based on pI values
Proteins migrate toward high pH end: protons are pulled off until balanced charge is 0 and protein stops moving

Question 15

Edman degradation

Answer 15

Method of protein sequencing
Phenyl isothiocyanate is added to proteins: N-terminus amino acid is released
Phenyl ITC + N-terminus amino acid is read at 254 nm and ID’d by HPLC to determine identity of amino acid
Done in succession to determine entire sequence

Question 16

Problems with Edman degradation

Answer 16

Not 100% accurate: after a while, chain doesn’t always release N-terminus amino acid or releases other amino acid
Works for chains of about 40 amino acids

Question 17

Mass spectrometry

Answer 17

Used to analyze protein mixture/sequence
Mass of molecules can determined if they are gaseous and charged
2 types: MALDI (matrix-assisted laser desorption ionization) and ESI (electrospray ionization)

Question 18

MALDI-TOF (time of flight) mass spectrometry

Answer 18

Protein is embedded in matrix and then shot with laser to make into gas, then ionized and accelerated through electric field
Lightest ions arrive at detector first
Solving for mass of peptide using library of peptide masses can be used to determine identity of peptide
Drawback: doesn’t tell primary structure

Question 19

Solving for unknown peptides using mass spectrometry

Answer 19

Chop up peptide into smaller pieces: bombard parent ions with inert gases to make smaller product ions
Pattern of dissolution can be used to piece peptide together

Question 20

Monoclonal antibody

Answer 20

An antibody that recognizes one epitope (structural site) on a protein

Question 21

Polyclonal antibody

Answer 21

A mix of antibodies that recognize multiple epitopes (structural sites) on a protein

Question 22

ELISA (enzyme-linked immunosorbent assay)

Answer 22

Enzyme-linked antibodies bind to target, allowing detection of target

Question 23

Western blotting

Answer 23

After SDS-PAGE, proteins can be transferred to a membrane and probed with antibody

Question 24

Peptide mass fingerprinting

Answer 24

1. Protein mix is run on 2-D gel electrophoresis
2. Single protein of interest is cut out of gel
3. Digestion of protein followed by elution
4. Protein is analyzed by mass spectrometry
5. Peptide masses are compared with theoretical masses of peptides from genomic database to find sequence

Question 25

X-ray crystallography

Answer 25

Crystallized protein is shot with x-rays
Diffraction pattern is based on atomic size and interference of waves
Electron cage can be reconstructed from diffraction pattern

Question 26

Resolution in x-ray crystallography

Answer 26

Depends on the wavelength of the x-ray: the smaller the wavelength, the easier to distinguish individual components

Question 27

NMR

Answer 27

Measure shift in magnetic spin (protons) at a certain frequency (resonance)
The nuclei’s environment will cause it to shift its spin at different frequencies (chemical shifts)