[3dem] How to make grids positively charged during or after glow discharge.
Paula da Fonseca
pauladf at mrc-lmb.cam.ac.uk
Wed Jul 3 13:46:21 PDT 2019
Hi Sacha and Bradford,
This is our version of the pentylamine treatment:
https://www.ncbi.nlm.nih.gov/pubmed/28580914
Certainly it should be done with suitable precautions...
Best wishes
Paula
On 03/07/2019 21:33, Sacha De Carlo wrote:
> Oui :)
>
> That’s the original but there more recent protocols maybe by Harris,
> with step by step procedures.
>
> Though the chemicals are poison and may not be used anymore; found on
> researchgate:
>
> "pentylamine or iso-amylamine" technique developed by Dubochet for the
> protein-free spreading of DNA was substituted, because these amines
> are poisons, with two others: by Robley Williams, 1977 ("poly lysine
> technique", this is mentioned above) and by Keller (''BAC technique",
> Vollenweider HJ, Sogo JM, Koller T. A routine method for protein-free
> spreading of double- and single-stranded nucleic acid molecules. Proc
> Natl Acad Sci U S A. 1975 Jan;72(1):83-7.). Both last methods work
> well and reproducibly.
> Good luck.
>
> Sent from my mobile device
> -----------------------------
> Sacha De Carlo, Ph.D.
> Business Development Manager EM
> Dectris.com <http://Dectris.com>
>
> On 3 Jul 2019, at 22:23, SCHMUTZ Marc (ICS)
> <marc.schmutz at ics-cnrs.unistra.fr
> <mailto:marc.schmutz at ics-cnrs.unistra.fr>> wrote:
>
>>
>> You mean that paper Sacha ?
>>
>>
>> A new preparation method for dark-field electron microscopy of
>> biomacromolecules1
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#aep-article-footnote-id1>2
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#aep-article-footnote-id2>3
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#aep-article-footnote-id3>
>>
>> Author links open overlay panelJacquesDubochet4
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#!>MichelDucommun4
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#!>MaxZollinger
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#!>EdouardKellenberger4
>> <https://www.sciencedirect.com/science/article/pii/S002253207180148X#!>
>>
>>
>>
>> Journal of Ultrastructure Research
>> <https://www.sciencedirect.com/science/journal/00225320>
>>
>> Volume 35, Issues 1–2
>> <https://www.sciencedirect.com/science/journal/00225320/35/1>, April
>> 1971, Pages 147-167
>>
>> regards
>>
>>
>> Marc
>> ------------------------------------------------------------------------
>> *De: *"Sacha De Carlo" <sacha.decarlo at dectris.com
>> <mailto:sacha.decarlo at dectris.com>>
>> *À: *"Ross, Bradford" <bradford.ross at botany.ubc.ca
>> <mailto:bradford.ross at botany.ubc.ca>>
>> *Cc: *3dem at ncmir.ucsd.edu <mailto:3dem at ncmir.ucsd.edu>
>> *Envoyé: *Mercredi 3 Juillet 2019 22:09:02
>> *Objet: *Re: [3dem] How to make grids positively charged during or
>> after glow discharge.
>>
>> You would need to read the old classics by people trying to look at
>> dna in negative staining: look for old protocols using pentylamine
>> (if I find them, I will share them with you) but I’m sure there are
>> some review papers too.
>> Best
>> Sacha
>>
>>
>> Sent from my mobile device
>> -----------------------------
>> Sacha De Carlo, Ph.D.
>> Business Development Manager EM
>> Dectris.com <http://Dectris.com>
>>
>> On 3 Jul 2019, at 20:06, Ross, Bradford <bradford.ross at botany.ubc.ca
>> <mailto:bradford.ross at botany.ubc.ca>> wrote:
>>
>> Hello Everyone,
>>
>>
>> I'm currently trying to image some lipid nanoparticles which have
>> a positive charge on their outer surface. Of course, since normal
>> glow discharge treatment typically yields a negative charge on
>> the grids, the particles are all sticking to the lacey carbon
>> support instead of remaining suspended in the vitreous ice layer.
>>
>>
>> I have heard that Magnesium Acetate can be used to switch the
>> charge from negative to positive after glow discharging, but
>> can't seem to find any protocols for exactly how to treat the
>> grids with it.
>>
>>
>> Does anyone have a protocol for the use of Magnesium Acetate, or
>> any other suggestions on how to flip the surface charge of the
>> grids to positive? Or other creative solutions I'm not thinking of?
>>
>>
>> Cheers,
>>
>> Bradford Ross
>>
>> Electron Microscopy Technician
>> BioImaging Facility
>> University of British Columbia
>> Cunningham Building Rm. 64
>> 2146 East Mall
>> Vancouver, B.C.
>> V6T 1Z4
>>
>> phone 604-822-6996
>>
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--
Paula da Fonseca
MRC Laboratory of Molecular Biology
Cambridge Biomedical Campus
Francis Crick Avenue
Cambridge CB2 0QH
UK
Tel: + 44 1223 267076
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