Cell fractionation Flashcards
1
Q
What is cell fractionation ?
A
Involves the seperation of cellular components
2
Q
What is the purpose of cell fractionation?
A
- Investigate protein trafficking and the re-localisation of proteins in response to stimuli
- Investigate post-translational modifications
- Reduce the number of proteins in a sample for proteomics
- Perform organelle-specific experiments
3
Q
What is the rate of sedimentation dependent on in centrifugation?
A
- Density of the particle
- Radius of the particle
- Viscosity of the medium
4
Q
What is differential centrifugation?
A
- Done at a low speed
- Sample is ‘pelleted’
5
Q
What is rate zonal centrifugation?
A
- Particles separated based on size
- Forms a density gradient (either continuous or discontinuous)
6
Q
What is isopycnic centrifugation?
A
Separation based on density
7
Q
What is the next step after the tissue is fractionated?
A
- Determine success of fractionation (protein concentration and yield)
- Calculate specific activity (increase or decrease in a particular fraction)
8
Q
What des cytochrome oxidase assay for?
A
- Measured using an oxygen electrode assay
- Assay for mitohondrial activity - consumprion of oxygen
9
Q
What does alcohol dehydrogenase assay for?
A
- Cytosolic protein
- Produces NADH which absorbs UV at 340nm
10
Q
What is Beer-Lambert’s law?
A
Absorbance (A) = constant x concentration (c) x pathlength (l)
