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Hi Andrei,
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<div class="">We have the EM GP but I have used the GP2 and hopefully this helps;</div>
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<div class="">1) My personal experience with the blotting sensor on the GP is that it fails- I didnt have much better experience with the GP2- however we held a course last week where a participant reported very good consistent results- but personally I cannot
recommend. </div>
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<div class="">3) This is subtly different if you plan front blot or back blot of the grids. The phrase I use when training users is ‘full, firm and flat’ contact- if doing back blotting this typically needs to be a shade ‘firmer’ in contact and benefits from
0.5 ul being on the back of the grid to ensure the liquid draws through the grid. </div>
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<div class="">The tweezers should not cause the grid to bend. Be aware- With the GP, the movement of the arm between the settings menu and main workflow is annoyingly slightly different-so optimise. You can check by running an actual cycle on the main menu
and you may have to adjust again- ours the actual motion tends to be 1-3 mm short, so it looks like the contact is too firm on the settings menu but just right when you actually run the cycle. May not be a problem with the GP2 but one to watch.</div>
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<div class="">4) My recommendation is always to position the grid further down towards the centre of the filter paper for precisely the reason you state, this helps to over come the wrinkling. I do also recommend replacing the filter paper fairly often (before
it becomes too wrinkly) this also improves reproducibility. </div>
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<div class="">5) I typically use 2.5-4s, ‘front blotting’ for SPA projects with 3ul. Front blotting tends to be a bit more reproducible in my hands, and I only use back blotting if I have a need (eg cells on grids, trying to concentrate filaments onto a grid)</div>
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<div class="">Hope this helps- good luck with your optimisation. </div>
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<div class="">For anyone interested in learning more on sample preparation and also data acquisition, you can check out the (presently remote) courses we are running alongside other partner sites in the UK <a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__astbury.leeds.ac.uk_facilities_wellcome-2Dmrc-2Dtraining-2Din-2Dcryoem_&d=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=ajUw8T5Tk31Hnw2msYTMk39Y6cJZZoWrDhQTMgR46utGxcjT6ZaJQ9WATpjMlmdJ&s=Opyqv5UQUZPBhnuTtoJF8sxddwgQiQssApfxS7aUqgU&e=" class="">here</a>. </div>
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<div class="">Best wishes,</div>
<div class="">Becky</div>
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<div>______________________________________________________________<br class="">
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Rebecca Thompson (She/her)<br class="">
Head of Faculty Biological Sciences Research Facilities<br class="">
Deputy Director, Astbury Biostructure Laboratory Electron Microscopy <br class="">
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University of Leeds<br class="">
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<div class="">On 4 Dec 2021, at 08:55, Andrei Moiseenko <<a href="mailto:postmoiseenko@gmail.com" class="">postmoiseenko@gmail.com</a>> wrote:</div>
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Dear all,<o:p class=""></o:p></div>
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I am trying to get reproducible results with Leica EM GP2. I would be very grateful if you could share any successful best practices on using this plunge freezer.<span class="Apple-converted-space"> </span><o:p class=""></o:p></div>
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Specifically:<o:p class=""></o:p></div>
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<span class="">1.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> <span class="Apple-converted-space"> </span></span></span>Do
you use the blotting sensor? In my case it fails very often and it seems that the results are better without it.<span class="Apple-converted-space"> </span><o:p class=""></o:p></div>
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<span class="">2.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> <span class="Apple-converted-space"> </span></span></span>In
case you use the blotting sensor, what range of “additional movement” do you use?<o:p class=""></o:p></div>
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<span class="">3.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> <span class="Apple-converted-space"> </span></span></span>In
case you use manual blotting, how do you set the horizontal blot position: just by touching the grid with the filter paper, or slightly bending it?<o:p class=""></o:p></div>
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<span class="">4.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> <span class="Apple-converted-space"> </span></span></span>How
do you set the filter paper vertical position: covering the grid only, just touching the forceps tips or higher (how much higher)? It seems that setting the higher position helps to reduce the effects of the filter paper crinkling.<o:p class=""></o:p></div>
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<span class="">5.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> <span class="Apple-converted-space"> </span></span></span>What
range of blotting time and specimen volume do you typically use for SPA samples at conditions specified?<o:p class=""></o:p></div>
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Any advice on using the device is very welcome.<o:p class=""></o:p></div>
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Best regards,<br class="">
Andrey Moiseenko<o:p class=""></o:p></p>
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Electron Microscopy Lab @ Biology Department<br class="">
Moscow State University<br class="">
<a href="mailto:postmoiseenko@gmail.com" style="color: rgb(149, 79, 114); text-decoration: underline;" class=""><span style="color: blue;" class="">postmoiseenko@gmail.com</span></a><o:p class=""></o:p></p>
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<o:p class=""> </o:p></div>
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