Dear all,
I was puzzled by my present TEM results: PTA staining showed a significant low frequency of AAV empty capsids compared with that of UA staining. I used the same sample and conditions (only different in staining reagents), and I really dont know why UA staining could show so many VP with black dot within (empty), however PTA just got many bright full capsids.
And I rarely noticed AAV empty capsid was measured using PTA as staining solution.
Does anyone know the possible reasons?
MANY THANKS!
Hi,
Most likely you have positive staining of the cargo of the capsids with UA as it also binds phosphate groups in DNA and RNA. So with the UA stain the bright capsids are the empty ones.
cheers,
Rob
Another reason is that PTA is a much larger molecule than UA, thus may not penetrate into capsid as readily. PTA (depending on pH) has also been shown to destroy viral particles if stained for longer than 30 seconds.
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