Lecture 11 and 12 - Biochemical methods. Flashcards
Stages in protein purification
1 - Sample homogenisation causing physical disruption of cells or tissues
2 - Differential centrifugation to isolate different cellular components
3 - Separation of proteins based on solubility, size, charge, hydrophobicity, and ligand affinity
Protein precipitation
Proteins can be precipitated by adding competing solutes such as ammonium sulfate ((NH₄)₂SO₄), acetone, or polyethylene glycol
Competing solutes: what characteristics should they ideally have?
Very soluble in water
Relatively non-denaturing
Easy to remove
Not too viscous/dense
Cheap and pure
Chromatography
Stationary phase - pigments at the bottom of the paper
Mobile phase - pigments move up the paper
Techniques for cell disruption
- Mechanical - blender blades disperse cells/tissues
- Liquid homogenisation - sheared through a narrow space by a homogeniser
- Sonication - high frequency and waves shear cells using a sonicator
- Freeze/thaw - ice crystal formation after continuous freezing through a freezer disrupts cells
- Manual grinding - Grinding through a mortar and pestle
What determines whether passive transport occurs?
Free energy of transport (ΔG)
If the value is positive, energy is needed to make movement occur
If the value is negative, energy is not needed to make movement occur (passive)
Affinity chromatography
Immobilised molecules with affinity for a protein are used to trap a protein and then, after the rest of the solution flows ahead, a competitor molecule elutes the protein, allowing the collection
Elution
The removal of a substance (usually in chromatography)
Usually by competition but can also be done by changing buffer conditions
Types of affinity chromatography
Immunoaffinity chromatography
Immobilised ligand chromatography
Lectin-based affinity chromatography
Immobilised metal affinity chromatography
Immunoaffinity chromatography
The antibody against protein of interest
Immobilised ligand chromatography
Substrate analogue or inhibitor binds enzyme or protein (e.g. heparin and heparin-binding proteins)
Lectin-based affinity chromatography
Lectins bind glycosylated proteins; elution is via the addition of sugars (e.g. N-acetyl glucosamine)
Immobilised metal affinity chromatography
metal ions (e.g. Ni2+) bind engineered recombinant proteins containing a poly-histidine tag at the N- or C-terminus.
Fusion proteins
Chimeric proteins engineered by the joining on the same polypeptide chain the products of two genes
Used for a variety of reasons, including affinity chromatography
PAGE
Polyacrylamide gel electrophoresis
Used to separate macromolecules
What can metal3+ elements do?
Bind phosphorylated proteins
SDS-PAGE
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (CH3-(CH2)11-SO4–NA+)
This molecule is a hydrophobic, dual ionic character detergent molecule
It forms complexes with denatured proteins at neutral pH and can be used to detect molecular weight
GST-fusion affinity chormatography
Glutathione S-transferase (GST)-fusion purified using immobilised glutathione GSH
What do micelles do in an aqueous solution
Behave as phospholipids would, forming a hydrophilic outer layer and hydrophobic inner layer
Assessment of sample purity
Purity - the more bands, the less pure
Abundance - the higher the band intensity, the higher the abundance
(Can only see marks after staining the gel with dye)
Protein purification table
Purification level indicated by the specific activity
What happens when proteins are at high/low pH
At high, the protein acts as an acid and becomes deprotonated
At low, the protein acts as a base and becomes protonated
Isoelectric point
The point at which a protein has an equal number of positive and negative charges
Isoelectric focusing
By creating a column with varying pH, proteins will naturally associate in the area where their isoelectric point is
Mass spectrometry
Are you being fr.
The molecule gets ionized and then fragmented. Fragments are put in locations based on their weight which is gathered through the use of a magnetic field
Polyclonal antibodies
Mixed populations of different antibodies which recognise different epitopes on the antigen
Monoclonal antibodies
Uniform population of antibodies which recognise the same epitope on the antigen
Immunoaffinity chromatography
Protein purification:
Monoclonal antibodies against the protein of interest
Adsorption:
Highly specific in favourable cases can be a one-step purification
Elution:
Tight binding can be drastic, often via pH change
Immunoblotting
?
Immunofluorescence
Protein labelled with a fluorescent molecule and then visualised with a fluorescence microscope
Actin filaments - red fluorescent protein (RFP)
Microtubules - green fluorescent protein (GFP)
Nuclei - blue fluorescent dye (DAPI) binding to DNA
Immunogold labelling
Antibody labelled with gold colloid particle and then visualised with an electron microscope
Passive transport across a membrane equation
ΔGₜ = RT x ln [substrate]ₒᵤₜ/[substrate]ᵢₙ
Centrifugal separation equation
a = ω2r
