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Hi Francesca,
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<div class="">We work with a range of specimens in buffers similar to the ones you describe and get nice ice with good particle contrast, so imagine you won’t have many issues. If your protein is on the small side or has significant disordered regions then
the salt concentrations may mean overall image contrast becomes a problem with downstream processing, but with most specimens we work with I don’t worry about 300 mM NaCl too much, if thats what the specimen is happy and stable in. </div>
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<div class="">Parameters seem to vary site to site, machine to machine and sometimes even month to month, but for us, moving the blot arm to ~212- 216 (get this so the grid makes firm flat contact with the filter paper, but grid does not start to bend), blotting
3-5 seconds is the range that gives good results, with the chamber at 8oc and 95 % nominal RH, for R1.2/1.3 holes (larger holes use smaller blot times). This is blotting so the meniscus directly hits the filter paper (not back blotting). We use Whatman no1
filter paper, and a range of glow discharge instruments but standard glow discharge in air, 30-90 seconds, HT 10. I would say these are the critical parameters, but email me offline and I can provide a full set if it would be useful :)</div>
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<div class="">There are so many parameters you CAN adjust when you are first optimising a new project it can be really tricky to work out what is critical for your system. If its a Leica EM GP thats already in use, asking the previous users what their (successful
or unsuccessful!) parameters are will give you a place to start. We also published a review (<a href="https://journals.iucr.org/d/issues/2018/06/00/ic5104/index.html" class="">here</a>) on sample prep which you may find useful.</div>
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<div class="">Hope this helps and good luck! </div>
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<div class="">Best wishes,</div>
<div class="">Becky</div>
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Rebecca Thompson<br class="">
Senior cryo-Electron Microscopy Support Scientist/Facility manager <br class="">
Astbury Biostructure Laboratory<br class="">
University of Leeds<br class="">
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Email<span class="Apple-tab-span" style="white-space: pre;"> </span><a href="mailto:r.f.thompson@leeds.ac.uk" class="">r.f.thompson@leeds.ac.uk</a><br class="">
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<blockquote type="cite" class="">
<div class="">On 3 Sep 2018, at 10:55, frangube <<a href="mailto:frangube@gmail.com" class="">frangube@gmail.com</a>> wrote:</div>
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<div class="">Dear all, <br class="">
I was wondering if anyone has experience in obtaining nice, thin ice by freezing a protein in 300mM NaCl (and 20mM Hepes pH 8, no detergent) using a Leica EM GP.
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If anyone has suggestions I would really appreciate it!<br class="">
<br class="">
Thanks in advance.<br class="">
<br class="">
Francesca<br class="">
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Dr Francesca Gubellini<br class="">
Unité Microbiologie Structurale<br class="">
Institut Pasteur<br class="">
25 rue du Docteur Roux <br class="">
75015 Paris<br class="">
France <br class="">
Tel: +33 (0)1.40.61.35.31<br class="">
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