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 2020-03-15 23:04:36