Biochemical Analysis Flashcards
(17 cards)
Stages in protein purification
- Sample homogenisation (allows molecular components to become available)
- Centrifugation to isolate different cellular components
- Separation of proteins on the basis of:
* solubility
* charge
* size
* hydrophobicity
* affinity for ligands
How can proteins be fractionated on the basis of solubility?
By adding competing solutes such as ammonium sulphate, acetone or polyethylene glycol = crude separation
These solutes compete with the proteins for the water in solution
What should competing solutes be?
- very soluble in water (up to 50% weight/volume)
- non-denaturing
- easy to remove from the proteins afterwards
- not too viscous or dense
- cheap and pure
Principles of dialysis
Separation on the basis of size
- Dialysis bag acts as buffer that only allows certain proteins to pass
- concentrated solution in the bag
- buffer outside
Describe passive transport in dialysis
Glucose moves out of the membrane to the lower concentration
∆G > 0
At equilibrium, glucose concentrations are equal inside and outside the membrane
Principles of chromatography
- pigments are loaded on the bottom of the filter paper (stationary phase)
- Organic solvent diffused up from the bottom (mobile phase)
- Pigments with different mobility are separated
What happens in chromatography?
- Sample is added to the stationary phase
- Flow of mobile phase takes the sample through the column
- Different substances separate and appear at the bottom of the column after different volumes have been added
What is ion exchange chromatography?
Elution of bound proteins from the column can be brought about by competition with increasing concentrations of NaCl
What is affinity chromatography?
- immobilised molecules with affinity for the protein is used to trap the protein of interest in the column
- the protein of interest binds and the rest leaves through the column
- or the protein of interest is eluted by adding a competitor molecule or changing conditions (e.g. pH or salt concentration)
Different types of affinity chromatography
- Immunoaffinity = antibody against protein of interest
- immobilised ligand = substrate analogue or inhibitor binds enzyme or protein
- lectin based = lectin binds glycosylated proteins and elution is via addition of sugars
- immobilised metal affinity = metal ions bind engineered recombinant proteins containing a poly-histidine (has good affinity to bind metals) tag at the N or C terminus
SDS-PAGE
- at neutral pH, SDS forms complexes with polypeptide chains in denatured form
- complexes are negatively charged as the amount of SDS molecules outweighs the number of charges on the protein chain
- each protein is converted to linear, denatured polypeptides in complexes with SDS
Disulfide bridges in SDS
- are not affected by SDS
- S-S bridges will affect SDS incorporation = changes in mobility
- connected subunits will move as a single chain so the bond MUST be broken
- reducing agents cleave S-S bonds = ß-mercaptoethanol, DTT, TCEP
Reducing SDS-PAGE
- Proteins are fully denatured
- Uniform SDS incorporation
- Keep relation between mobility and molecular weight
- Seperate subunits to their individual chains
What is protein purity?
Separated as bands vertically on a lane
What is abundance?
Band intensity
Identification of proteins by mass spectroscopy
- Band/spot is removed from gel
- The isolated protein is cut using proteases
- Peptide seperation by chromatography
- Each peak is investigated by M/S
- Observed set of peptide masses is compared with predicted = protein identification
Immunofluoresence
- antibody is labelled with a fluorescent molecule and then visualised with a fluorescence microscope
- actin filaments seen as red
- microtubules are labelled with GFP
- nuclei labelled with blue dye
