we saw this slide, a summary of the process,
what do want to achieve:
- separate components of the tissue
- last tube has:
- cytosol of
what is in the last tube?
- insoluble and soluble components
- insoluble → ribosomes, viruses, other macromolecules
- soluble
- the scheme here, is to get from liver tissue, if we’re interested, we could be interested in sub-cellular compoenetns (mitochondria, nucleus, peroxisomes, etc)
- or we could be interested in the soluble components present in the supernatant
- soluble → not particles, cannot separate them with centrifugation
what is the centrifugation technique?
- differential centrifugation → main function\use of it is to separate sub-cellular components or enrich ?
going back to supernatant, what do you expect?
- all soluble materials that can’t be to be removed
- glucose
- salts
- ions
- proteins
- lipids
- tld whatever is soluble; cell has thousands of cellular components,
e.g liver cell
- every metabolism that goes thru will go thru the liver cell (main energy production organ)
- oxidation of glucose → cytoplasm of liver cell etc
- liver is the main organ in biochemical processes, so we expect alot of compoentns
suppose nowe im interested in a partciular protein from the liver tissue, and there are many other components in the supernatatn
- how do i get rid of these metabolites before i even proceed to the next step?
- we said in supernatant, whatever in it is soluble, no way to separate them just by centrifugation
- what should we do?
- filtration (can be used, but depends on what we want) → doesnt remove small molecules if we use filter paper, we need another type of filtration (only if macromolecules can be trapped
- proteins rquire ultra-filtration, then we can possibly separate the proteins
- dialysis (most common)
- now we are left in proteins, how maany do you expect? alot
- how can you separate the proteins
- electrophoresis
- chromatography (this is the ideal method)