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The case of the punky Sf9 cells - (Jun/20/2011 )

Hi everyone!
My student had cloned and expressed a capside protein from a bovine retrovirus (His-tagged) in the baculovirus system. She has got the expression correctly (checked by WB) but the cells look "punky". When we stained the cells with a FITC conjugated anti-His Mab (MACS Molecular), the cytoplasm and the "spikes" get fluorescent (see attached picture). We then stained with Alexa Fluor 488 phalloidin, and it looks the same, but red, with long convergent structures organized from the center of the cells, suggesting some kind of association of the viral protein with the cytoskeleton. Had anyone seen something like this before? Any comment or suggestions?
Attached File

-clutz-

Adherent or suspension culture?

One thought is that capsid proteins are often self-assembling - perhaps the structures you are seeing with the "spikes" are self-assembled protein chains.

-bob1-

bob1 on Tue Jun 21 01:14:42 2011 said:


Adherent or suspension culture?

One thought is that capsid proteins are often self-assembling - perhaps the structures you are seeing with the "spikes" are self-assembled protein chains.



Hi bob1! thanks for your comment
Cells are adherent and yes, self assembly is one possibility.I am also aware that capsid proteins from other viruses also interact with cytoskeleton components in some way, but the kind of protrusions observed are more like dendrites, and not such a long spikes...anyway the protein is under a strong promoter so it is over expressed. We also observed tha t the cytoplasmic membrane was intactů
I was wondering if someone has observed something like this with other proteins.

-clutz-