Svedberg calculation exparament.

[havery01]

A newly discovered organelle is centrifuged in 30-60% discontinuous sucrose desity gradients made with pH 4, 6, and 8 buffers. The bands produced are examined, Svedberg values calculated, and the material in each band examined for activity. The results are:

buffer pH, number bands and Svedberg values, Functional
4 70S yes
6 58S yes
8 22S, 20S, 4s 22S fraction is functional but at ~25%

a)provide the instructions to make 200 ml of 60% sucrose in 0.85% NACl and 0.1 M Tris.

b) what information about the organelles structure can you infer from the Svedberg data from the three centrifugations?

c)why does the organelle have different Svedberg values following centrifuging at pH4, pH 6, and pH 8?

d)explain why the Svedberg values at pH 8 do not equal the Svedberg value at pH 4 since the same organelle was centrifuged?

Me: i know how do do svedberg calculations, but what i dont understand about this question is how they can have different svedberg values while using the same buffer and the same organelle. is it calculating the svedberg values for the different bands that showed up during this experiment? As for question b) i think you can infer from the structure that the organelle cant have too many proteins because only one band was produced in two of the electrophoresis runs. I dont completely understand what question a) is asking.

[JackBean]

a) just write how to prepare that solution…

b) & c) pH 4 – 1 band -> pH 6 – 1 (smaller) band -> pH 8 – 3 bands
The Svedberg value is dependent both on size AND shape of particle!
so, every band means particles of one size and one shape and because you’re always centrifuging the same thing, it cannot just disappear or show up, it must be there all the time. So, different pH probably causes disagregation of the organelle

d) you should know that…

[Author]

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