Protein - Analysis and Sequencing Flashcards
How are we able to distinguish/separate proteins?
Polypeptide diversity
There are an unlimited amount of theoretical possibilities of polypeptides - in reality they are limited in size and composition
Why do we need to purify proteins?
The design of drugs that inhibit/alters the function of a protein,
The re-design of proteins with novel properties
Use of enzymes in the bio-pharmaceutical industry
Understanding of the causes of disease
What are the two broad methods of purification?
Protein is common/abundant:
Use a convenient and well understood species e.g. rubisco
Protein is expressed at low levels and very specific:
Isolate from specific organism or express in each to handle species e.g. HIV proteins
Both: require breaking open the cell and fractionate by different methods
What factors do we need to control to keep proteins stable?
pH - buffers prevent denaturation
Temperature - most denature at high temperatures
Protein purification tends to be at 0 degrees
Degradative enzymes (proteases/nucleases) - can be released when cells are disrupted to liberate the protein
Adsorption to surfaces - proteins can denature by contact on air-water
Give an overview of the fractionation process of purification of proteins?
- Break up the cell either mechanically or chemically (acquire the homogenate)
- Remove most of the contaminating material by differential centrifugation (acquire the pellet and supernatant)
- Isolate the protein by column chromatography - the unique combination of properties of the protein (e.g. mass, size, charge, shape) enable its separation
- Check the purity by electrophoresis
How can we track proteins through the process of purification?
Quantified by assays
Absorbance - some are coloured and others absorb UV-ligth (absorbance is proportional to amount of protein)
Visualisation - Using dye
Activity - identifying characteristic of an enzyme
What are some of the most sensitive assay techniques?
Immunoassays that use antibodies in response to an antigen
RIA - Radioimmunoassay
ELISA - Enzyme-linked immunosorbent assay
Describe ELIZA?
- Immobilise the first antibody on a solid support
- Incubate with a protein containing sample
- Add a second antibody that is covalently linked to an assayable enzyme
- Wash and assay enzyme activity
The amount of substrate converted to product indicates amount of protein present
What are the physiochemical properties of proteins to distiguish during purification procedures?
Solubility - salting out
Ionic charge - ion exchange chromatography, electrophoresis & isoelectro focusing
Polarity - hydrophobic interaction chromatography
Size - gel filtration chromatography & SDS-PAGE
Binding specificity - Affinity chromatography
Describe salting out?
As more salt is added, the solubility of the protein decreases = salting out
The salt allows the proteins to clump together acting as a barrier between different charged groups
Centrifuge to acquire the correct protein
Proteins precipitate at different concentrations of ammonium sulphate
Good first purification step
What is chromatography?
There is a mobile and stationary phase in efforts to separate molecules
Solutes pass through the column, interacting with the stationary phase and are retarded to differing extents
What does ion exchange chromatography do?
Property - Ionic charge
Charged molecules bind to oppositely charged groups chemically linked to a solid matrix (e.g. cellulose or agarose)
Amino acids have different charges due to their R groups and the pH they are in = different isoelectric points
What are the two types of ion exchange chromatography?
Cationic exchange - a cation protein will bind to anionic groups in the cation exchange resin - CM: carboxymethyl
Anionic exchange - an anionic protein will bind to cationic groups in the anionic exchange resin - DEAE: diethylaminoethyl
How can the charge of a protein be affected within ion exchange chromatography?
pH alters the the charge and the position of the pI in relation to pH changes the binding
Altering the pH at extreme ends could denature the protein, so changing salt concentration e.g. increasing NaCl - can displace proteins via competition through higher charge densities
Describe hydrophobic interaction chromatography?
It purifies non-polar molecules
The matrix is substituted with octyl/phenyl groups
At high salt concentrations non-polar groups interact with the hydrophobic groups in the matrix
What does gel filtration/size exclusion chromatography do?
Property - Size
Molecules are fed into a column of gel beads containing pores
Small molecules - diffuse through the pores (takes longer)
Larger molcules - go around the beads (come out first)
How can we control gel filtration chromatography?
Different size beads and therefore pores are used to allow different sized proteins to be separated
There is a resolution range for the size of the pores to determine which proteins can be separated
Within this range, there is an inverse linear relationship between the volume at which a protein elutes and the log of the Mr
If we mimic the native conditions of a protein we can work out it’s native size or its quaternary structure
What are the elutes and graphs in gel filtration chromatography?
The elute - as they come out if there is 3 for example: void - partial interaction - total interaction
On an elution graph:
Before the peak = high molecular mass contaminants
After the peak = low molecular mass contaminants
What does affinity chromatography do?
Property - binding specificity
Adding a chemical tag/ligand to a protein e.g. histidine
The tag binds to a resin and we can elute the protein by altering the pH or ionic strength
What is electrophoresis?
The migration of ions through an electric field
The electric field is passed through a gel - normally polyacrylamide
This forms a mesh and acts as a molecular sieve
Called PAGE - polyacrylamide gel electrophoresis
What are the types of electrophoresis?
Native PAGE
SDS PAGE
Isoelectric focusing
Describe Native PAGE?
Property - Size and Charge
Size - smaller proteins move faster than larger proteins
Charge - proteins with a larger charge move faster that proteins with smaller charge of the same sign
Negative to positive electrode, so pH is adjusted so proteins are negative (move down)
How can different proteins be affected in native PAGE?
If a protein is quaternary it will remain in tact - not separate
Proteins of a similar size but different charges - move different distance
Describe SDS PAGE?
Property - size (smaller = faster)
Sodium Dodecyl Sulphate (negatively charged detergent) binds 1 per 2 amino acids
It breaks the non-covalent structures = denaturation subunits/quaternary structure
The R groups become masked = equal charge across whole protein
Separation only depends upon the sieving effect of the acrylamide gel
What else can be used in SDS PAGE?
Addition of reducing agent allows presence/absence of disulphide bonds to be identified (as many quaternary structures are stabilised by these)
Mercaptoethanol reduces disulphide bonds in proteins
e.g. antibody split into its heavy and light chains
What can we deduce from SDS PAGE?
The relative mobilities of proteins vary approximately linearly with the log of their molecular mass
Low mass proteins elute first - the smaller the mass the greater the mobility
Single band by PAGE = pure protein
What is isoelectric focusing?
Property - charge
Proteins are poured along a gel with a pH gradient built in and the proteins are place anywhere within
Each protein will gain / lose protons according to the pH of the liquid phase in what its in
They stop moving when they reach a pH in the gel when their charge becomes zero – at their iso-electric point (pI)
Proteins of a similar size with different pI values will move different distances
As gel electrophoresis is slow what can be used instead?
Capillary Electrophoresis
Thin capillary tubes, rapidly dissipate heat and can use high electric fields - reducing separation times to a few minutes
What is 2D gel electrophoresis?
Isoelectric focusing combined with SDS-PAGE
The sample is subjected to IEF in one direction and then subjected to SDS-PAGE in the perpendicular direction
Useful for proteomics
How is electrophoresis visualised?
If radioactive - autoradiograph
If antibody present - immunoblotting or Western blotting
What does ultracentrifugation do?
Separates macromolecules by mass
Svedbergs (S) - sedimentation velocity per unit of centrifugal force
Adding CsCl = forms a density gradient under high fravitational field
