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ultracentrifugation

ok, I have 2 questions thats based on the process of ultracentrifugation, here they are:

The homogenate and supernatant contain an isotoic buffer.

a) explain why a buffer is used

b) explain why th buffer is isotonic


thanks
Reply 1
Buffer: Makes sure the enzymes are not denatured
Isotonic: Makes sure that no organelles burst through gaining water by osmosis

It's also cold to stop the activity of the enzymes.
mchammer
a) explain why a buffer is used


Keep the suspension at cellular pH?

mchammer
b) explain why th buffer is isotonic


Stop organelles from lysing/chaning shape/density?
Reply 3
thanks but I dont get the bit about activity of enzymes? since when were they involved?
Reply 4
Enzymes are in cells. They're very organised and when the cell is rather brutally cut up I imagine they mix with things and they could result in things being broken down. If you put them in a cold isotonic buffer then they have less kinetic energy which means less enzyme substrate complexes being formed and thus the activity of the enzyme being reduced. If your exam board doesn't pick up on it being cool, then I assume you don't need to know this.
Buffer doesn't affect kinetic energy really. All it does it keeps the homogenate at a cellular pH, approx. 7.4 (dependent on cell type). This will stop lysis and stuff.

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