I have a couple of questions about this protocol1) You centrifuge...

protocol Protocol: Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin
I have a couple of questions about this protocol

1) You centrifuge the flagellar filaments at 200,000 x g and then at 20,000 x g. What purpose do these two spins serve and why is there such a large difference between the speeds? If 20,000 x g is sufficient to pellet the filament
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Hiroyuki Hirai Author Answered Mar 15, 2020

Nagahama Institute of Bio-Science and Technology

1)
Flagella filaments of Acidovorax avenae can be pelleted by centrifugation at 10,000-20,000×g.
But, flagellin monomers and low polymeric filaments cannot be pelleted by this centrifugation.
Because we wanted flagellin monomers with PAMP activity, we performed centrifugation at 200,000×g.
However, the 200,000×g pellet also contained proteins other than flagellin protein.
We wanted to remove these proteins.
When re-suspended solution of the 200,000×g pellet centrifuged at 20,000×g, many proteins existed in the supernatant.
Thus, this section is an operation to remove many proteins.

2)
Plants recognize the monomeric flagellin as PAMP.
Because final pellet had the PAMP activity, we assume that this pellet contains monomeric flagellin.

Sincerely yours,
Hiroyuki Hirai
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