Tools of Biochemistry Flashcards
Proteomics
The study of proteins produced by the genome
Differences between proteins and genome
- Proteins are dynamic, whereas genome is fixed
- # and type of proteins change in each cell, whereas genome is fixed
- Proteins can change within cell under some conditions, whereas genome can’t change
- Proteins interact, whereas genome is non-interactive
Ways to study a protein
- Sequence it
- Run an assay that measures protein function
- Quantitate protein (specific activity= enzyme activity/total protein)
- Isolate protein from cells or tissue
- Solve its structure
- Identify its temporal and spatial environment
Using color to analyze proteins
Product has color; reactant does not
Differential centrifugation
Centrifuge at progressively higher speeds to separate out heavier materials of cell from lighter ones
Can be used to isolate spatially separated proteins
Salt precipitation
Salt draws water away from protein
Without water to solubilize, protein becomes insoluble
Dialysis
Semi-permeable membrane retains large molecules but lets small molecules into solution
Gel filtration chromatography
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
Ion affinity chromatography
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
Affinity chromatography
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
Electrophoresis
Proteins migrate on charged gel based on their size
Large friction due to large size or shape: less velocity and less movement
Ingredients for gel electrophoresis
- Polyacrylamide gel matrix
- Buffer to hold uniform negative charge
- Denaturing agents (beta mercaptoethanol or heat) to disrupt cysteine bonds
- Anionic detergent (sodium dodecyl sulfate) to give proteins negative charge
2D gel electrophoresis
Proteins are separated according to size and isoelectric point
Good separation of complex protein mixtures
Isoelectric focusing
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
Edman degradation
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
Problems with Edman degradation
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
Mass spectrometry
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)
MALDI-TOF (time of flight) mass spectrometry
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
Solving for unknown peptides using mass spectrometry
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
Monoclonal antibody
An antibody that recognizes one epitope (structural site) on a protein
Polyclonal antibody
A mix of antibodies that recognize multiple epitopes (structural sites) on a protein
ELISA (enzyme-linked immunosorbent assay)
Enzyme-linked antibodies bind to target, allowing detection of target
Western blotting
After SDS-PAGE, proteins can be transferred to a membrane and probed with antibody
Peptide mass fingerprinting
- Protein mix is run on 2-D gel electrophoresis
- Single protein of interest is cut out of gel
- Digestion of protein followed by elution
- Protein is analyzed by mass spectrometry
- Peptide masses are compared with theoretical masses of peptides from genomic database to find sequence
X-ray crystallography
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
Resolution in x-ray crystallography
Depends on the wavelength of the x-ray: the smaller the wavelength, the easier to distinguish individual components
NMR
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)
