[3dem] negative staining artifact

[Kenton Arkill]

I always filter my UA. 22um seems to work.
Kenton

On 25 Sep 2017 10:27, "Reinhard Rachel" <
Reinhard.Rachel at biologie.uni-regensburg.de> wrote:

> Dear Linda,
> besides the points discussed already, I am curious to learn why you use
> copper
> grids with formvar, instead of using grids purely coated with carbon films
> (prepared by evaporation onto mica, floating off, and then depositing the
> c-film onto the grids). Formvar can be nice, can be nasty.
> Second, I wonder whether it is your sample what we are looking at. Does it
> contain "phosphate-containing compounds" like PBS, ribosomes or remnants,
> etc
> etc? all PO4-containing substances - bio or not bio - will "attract"
> Uranylacetate. Many carboxyl-groups do attract Uranylacetate. Think about
> the
> positive staining effect of DNA, ribosomes, P-lipid-membranes, etc etc.
> How "pure" is the sample? What is the pH of the sample? and did you wash
> for a
> fraction of a second with bi-distilled water? we often do this - and hope
> the
> best that the sample survives. It may help.
> just my 2 cents ... kind regards - Reinhard
>  ============================
>
> > I believe its your stain precipitate . I too had this problem once (my UA
> > was old). I used a mild centrifuge rotation and took the supernatant for
> > staining and it worked. I hope this will solve your issue.
> >
> > Regards,
> >
> > Subhomoi B.
> >
> > On Mon, Sep 25, 2017 at 12:31 PM, Johanna Höög <johanna.hoog at gu.se>
> wrote:
> >
> >> Hej Linda!
> >>
> >>
> >>
> >> I had similar precipitates a while ago and it took me forever to find
> the
> >> source. For us, it was that the oil filter on the glow discharger that
> >> needed to be changed. Try just putting an empty grid in the glow
> discharger
> >> and then have a look at it to exclude this possibility.
> >>
> >>
> >>
> >> Good luck!
> >>
> >>
> >>
> >> Johanna
> >>
> >>
> >>
> >> *From: *3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Linda Sandblad
> <
> >> linda.sandblad at umu.se>
> >> *Date: *Sunday 24 September 2017 23:27
> >> *To: *"3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> >> *Subject: *[3dem] negative staining artifact
> >>
> >>
> >>
> >> Hi all experts!!
> >>
> >> I know, this do not look nice, but I would like to hear your opinion
> about
> >> this artifact. Tiny dark spots covering both sample and background, do
> you
> >> know what it is, were it comes from and how to avoid it? We have this
> bad
> >> background time to time from different labs…. It is a “normal” negative
> >> staining protocol: copper grids, formvar, carbon, glow discharge, UA.
> >>
> >> I’m happy for your suggestions, Best Linda
>
>
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