L11+12- Methods- protein purification Flashcards
What are th 5 lysis methods to physically disrupt cells?
- Mechanical- blender, polytron- blades mash up cells
- Liquid homogenization 0 shove cells through narrow space
- Sonication- High frequency sound wave shear cells
- Freeze/thaw- ice crystal formation disrupts cells
- Manual grinding
What is the formular for centrifugal force?
F= - Ω^2 r
Where Ω is the angular velocity (radians/sec)
which is 2πrpm/60 (rpm=revs per min)
r=radius in metres
What do you have to do to the value of centrifual force to get it in g?
Divide by 9.8
F/9.8 = xg
What usually homogenates first in centrifuge?
Nuclei, then mitochondria
What has more effect on centrifugal force? speed or radius?
Speed.
How can proteins be precipitated?
Bu adding competing solutes such as ammonium sulphate and polyethylene glycol. (precipitates then removed by centrifugation)
What 5 characteristics are competing solutes ideally?
- Very water soluble
- non-denaturing proteins
- easy to remove from protein afterwards
- not too viscous or dense
- cheap and pure
How can particles be crudely separated on basis of size?
Dialysis. Partially permeavle membrane. Smaller things will leave dialysis bag to water by osmosis.
How can particles be finely separated on basis of size?
Size exclusion chromatography.
Small molecules can enter the beads. Larger molecules can’t enter beads so flow down between them.
How can we separate on basis of charge?
Ion exchange chromatography. Have negatively charged beads. Postive proteins will bind to the beads. Negative proteins flow through. At the end add increasing concs. of NaCl to out compete the bound proteins and slowly they will come out.
How do you use affinity chromatography?
- Immunoaffinity chromatography
- Immobalised ligand chromatography - e.g. substrate analogue of an enzyme
- Addition of affinity tages to recombinant proteins (widely used method)
- Lectin-based affinity chromatography- binds glycosylated proteins. Elution is with sugars such as N-acetyl glucosamine.
How does elution happen?
Usually by competition but can also involve disruption of interaction by changing buffer conditions. (eg salt conc, pH)
What is SDS used for?
To denature and add uniform charge to proteins so that they move in PAGE relative to size.
What is isoelectrofocussing?
Seperation of proteins using high pH (-) at one end and low pH (+) at the other. The protein stops when it reaches its isoelectric point.
What can mass spectrometry show us?
Fragment mass/charge ratios. Can use the fragments to work out which proteins are present.
