[3dem] How to make grids positively charged during or after glow discharge.

[Paula da Fonseca]

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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