17 - Protein Detection Flashcards
C-reactive protein
Activates immune system. Triggered by cytokines and proteins
Why detect proteins
As a marker of inflammation and to detect pathology (e.g. myocardial infarction).
4 levels of protein structure
Primary (amino acids), Secondary (beta sheet and alpha helix), Tertiary (polypeptide chain) and Quaternary (assembled subunits)
Which proteins detected to indicate myocardial infarction
Contractile proteins (Troponin 1 and T)
Chromatography column
Solid porous matrix (“stationary phase”)
Mobile phase
Proteins sample that flows through chromatography column
Which proteins elute first
Proteins that move quickly (interact less well with column)
Which proteins elute last
Proteins that move slowly (interact more with column)
Fractionation
Collection of effluent at different stages. Different fractions have different properties
Eluent
Substance that goes through column chromatography
Effluent
Substance that comes out of column chromatography
Size exclusion chromatography
Column made of porous beads. Larger proteins do not adhere to column (therefore move quickly and elute earlier). Small proteins adhere to column (therefore move slowly and elute later)
How do proteins have different charges
The functional groups of individual amino acids confer an overall charge to the protein
What can change the charge on the AA side chain
pH. Acids lose/gain H+, bases lose/gain OH-
Ion exchange chromatography
Column is made of charged gel beads. If stationary phase is negatively charged, proteins with negative charge will move faster and elute earlier.
Issue with ion exchange chromatography
proteins with different charge to stationary phase will not elute out and therefore become stuck
How to fix issue with ion exchange chromatography
Add salt (charged ions). Ions outcompete protein for binding to column
Cation exchange
negative column, positive samples of interest
Anion exchange
Positive column, negative samples of interest
Affinity chromatography
Column made of beads with a molecule (ligand) to which the target binds. Target protein binds, non target are eluted.
how to elute target protein in affinity chromatography
With solution of ligand. Target protein will bind to free floating ligands instead of those attached to column
Other forms of affinity chromatography
Antibody - antigen
Avidin - biotin
Nickel - Histidine tag
How to elute affinity chromatography that doesn’t contain ligands
high pH dentures the proteins so they no longer bind
SDS-Page
SDS (detergent, denatures proteins, gives uniform -‘ve charge), polyacrylamide plastic (same function as agarose but seperate much smaller molecules). Larger proteins migrate less far
advantages of SDS-PAGE
Quick, easy and inexpensive
Disadvantages of SDS-page
Visualise proteins with stain (small amount detected), does not identify proteins, many proteins have similar mass, unable to tell how many proteins a band shows
Charge when pH is less than pKa
positive (protonated)
Charge when pH = pKa
No charge
charge when pH is greater than pKA
Negative charge (deprotonated)
Isoelectric focusing
Separates proteins by isoelectric point. Protein only moves through gel when it is charged. Reaches point in strip where pH = pI (protein has no charge, will not migrate further)
2D electrophoresis
Separates proteins by isoelectric point and size. Solves problem of co migration in SDS-PAGE
Western blots (immunoblots)
Transfer proteins from polyacrylamide gel to a membrane. Allows immobilisation of the protein which allows antibodies to interact with the protein
Components of Western blots
- Primary antibody recognises antigen
- Secondary antibody recognises primary antibody (enzyme conjugated)
- Enzyme allows detection (catalyses reaction to produce luminescence)
Enzyme Linked Immunosorbent Assay (ELISA)
Use antibodies to recognise specific antigen (protein of interest)
Direct ELISA
- Proteins are immobilised to a plate
- Antibody binds to target protein and is conjugated to an enzyme
- Enzyme produces colour product
- Less specific
Indirect ELISA
- Primary antibody binds to target protein
- Secondary antibody binds to primary antibody
- Secondary antibody is conjugated to an enzyme which produces a coloured product
Sandwich ELISA
- Capture antibody bonded to plate and binds to target protein
- Primary antibody binds a different epitope on the target protein
- Secondary antibody binds to primary antibody, conjugated to enzyme to detection
Which protein side chains absorb light
Aromatic group
High Performance Liquid Chromatography
- Column usually C18 (silica) which is hydrophobic.
- Sample dissolved in water and non polar solvent.
- Hydrophobic proteins in sample will bind to C18.
- Hydrophilic proteins elute.
- Change solvent over time to be more hydrophobic to elute the “stuck” hydrophobic proteins
- Achieves gradient of elution (hydrophilic –> hydrophobic)
Mass spectrometry
- Measure mass of compounds with great accuracy.
- Compound ionised
- Charge attracts it to detector
- Arrive at detector at different times based on mass to charge ratio
- Small mass to charge ratio and faster, large mass to charge ratio slower
- Also fragments peptides and measures products of fragmentation
Mass fingerprint
Two different proteins with the same mass will produce different peptides
How is protein prepared for mass spectrometry
Digested with proteolytic enzyme
Advantages of mass spectrometry
Generate huge amounts of detailed data
Disadvantages of mass spectrometry
- Expensive, complex machines needed
- Data can be difficult to interpret
- Difficult sample preparation
