Isolating & Analyzing Proteins 8 Flashcards
Subcellular fractionation
-separation of organelles based on their physical properties
Protein separation by size
- size exclusion chromatography
- smaller molecules enter porous beads (elute later)
- larger molecules cannot enter beads (elute earlier)
- column can be calibrated to provide a molecular weight estimates of an unknown.
- uses: looking for natural products
- specific activity/medicinal properties of certain proteins
- changing buffer
Protein separation based on charge
- ion exchange chromatography
- charged groups of protein bind to a charged resin, exulted with increasing salt
- protein mixture added to column containing cation/anion exchangers
- proteins move through he column at rates determined by their net charge at the pH used.
- revered phase chromatography
- beads contain non-polar groups that bind to protein hydrophobic patches (elated using less polar solvent)
Affinity chromatography
- most discriminating property of a protein (its function) can be used for isolation … very specific
- protein can bind to an immobilized specific ligand, substrate
- protein then eluded using ligand solution
Affinity tags
- affinity tags can be added to recombinant protein for expression and purification
- affinity tags essentially allows the protein to bind to a specific ligand so that it can be found more easily, and is then separated during elution
Ni2+ with affinity chromatography
- A nickel affinity tag can be used to bind a ligand
- the protein is then eluted using imidazole
- relies on the coordination of histidine with immobilized Ni2+
SDS-PAGE
- electrophoresis is the separation of charged molecules in an electric field
- most common method is SDS polyacrylamide gel electrophoresis (SDS-PAGE)
- due to sieving the gel, SDS-protein micelles are separated by size (MW).
- smallest proteins/peptides migrate the fastest toward the positive electrode
- reducing agent is usually added to cleave the S-S bonds and resolve individual polypeptide chains
- SDS denatures all proteins so they all unfold and have a neg charge
2D-PAGE
- separation by both pI and size can reveal hundreds of proteins and disease related differences in protein levels
- the proteins settle in the gel based on their isoelectric point
Protein sequencing by mass spectrometry
-determines mass:charge ratio
-start with protein of interest
-digest the protein with protease to form peptides
-trypsin cleaves peptide bonds at Lys or Arg
-peptides are released by tryptic digestion and their masses are measured using mass spec
-exact mass of fragments are screen and compared to other peptides
-matched with corresponding gene
OR
-each peptide can be further fragmented at peptide bonds and then measured, then can be used to construct partial amino acid sequence
-used to identify gene or provide means for cloning the gene
Dynamic range of protein analyses in plasma
- the potential: plasma contains multiple protein/peptide biomarkers that are diagnostic for disease and respond to therapy
- the problem: these consist of over 500,000 chemically distinct peptide and protein species, varying in amount by >10 orders of magnitude
X-Ray crystallography
- the 3D arrangement of atoms in a protein can be deduced by measuring the diffraction of X-rays in a protein crystal
- resolution of <1A is possible, providing detailed information on conformation, enzyme mechanism, and binding
- 137,000 protein structures now available in the Protein Data Bank and about 90% are from X-ray crystallography
-X-rays through crystal create a diffraction pattern, and is then phased into an electron density map, which can then help to identify AA for protein based on structure
Protein NMR
- nuclear magnetic resonance spectroscopy measure the environment of nuclei with spin
- the resonance frequency of nuclei in a strong magnetic field can provide information on nearby atoms (through bonds or space)
- signals can be assigned to specific atoms in a protein, providing an ensemble of 3D structures
- NMR can characterize protein motion (dynamics)
- Take AA sequence
- Assign spin signals to H atoms
- Identify pairs of spin atoms close in space
- Final structure can be determined
Cryo-electron microscopy
- Cryo-EM has increased in recent years due to technological advances in detectors and sample preparation
- excellent for large proteins and complexes, more limited for smaller proteins
- a beam of electron is fired at a frozen protein solution.
- the emerging scattered electrons pass through a lens to create a magnified image on the detector, from which their structure can be deduced
