<html><head><meta http-equiv="Content-Type" content="text/html; charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; line-break: after-white-space;" class="">Hi Sasha,<div class=""><br class=""></div><div class="">To add to what Rajiv said, it’s not clear from your image that there is even GO coating the hole that you are imaging. If have you a lot of GO on your grid, then you should see lines in your images representing the edges of GO sheets (see attached image for an egregious example). If you don’t have much GO on your grid, an atlas of a square should indicate that holes appear different (GO covered areas are usually darker) depending on the presence/absence of GO.</div><div class=""><br class=""></div><div class="">Best,</div><div class="">Ryan</div><div class=""><br class=""></div><div class=""><img apple-inline="yes" id="B2946F1D-F330-4FEB-ACF5-3B4137BBD39A" width="299.5" height="424.5" src="cid:57DDFCD2-B950-458B-A567-202A0A0A9BB9" class=""><br class=""><div><br class=""><blockquote type="cite" class=""><div class="">On Oct 10, 2022, at 4:36 PM, Rajiv Ranjan Singh <<a href="mailto:rajssai@gmail.com" class="">rajssai@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div dir="ltr" class="">Hey Sasha,<div class=""><br class=""></div><div class="">Try H2, O2 Plasma Cleaning for 30S in each case. If you see good particles in Negative stains. </div><div class=""><br class=""></div><div class="">I hope you are doing plasma cleaning before coating GO, if not then try following protocol from Eva Nogales lab.</div><div class=""><br class=""></div><div class="">Best,<br clear="all" class=""><div class=""><div dir="ltr" class="gmail_signature" data-smartmail="gmail_signature"><div dir="ltr" class=""><div class="">Rajiv Ranjan Singh</div><div class=""><br class=""></div></div></div></div><br class=""></div></div><br class=""><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Tue, Oct 11, 2022 at 4:47 AM SSS Sasha <<a href="mailto:sss_jiandan@outlook.com" class="">sss_jiandan@outlook.com</a>> wrote:<br class=""></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div class="msg1457054943890267078">
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Dear all,</div>
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I was trying to freeze cryo EM grids for a protein ~ 0.73MDa under several conditions, but it didn't work well. I make a brief summary here. Does anyone have any suggestions? Thanks in advance!</div>
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Holly grids CF-1.2/1.3-4Cu-50: plasma cleaning for 10s using O2,Ar2 Protein can't go in to the hole</div>
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Lacey carbon 400mesh Cu: plasma cleaning for 15s using O2, Ar2 Contrast is quite low, hardly see the particles</div>
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GO on Quantifoil: <span style="background-color:rgb(255,255,255);display:inline" class="">plasma cleaning for 15s using H2, O2 Protein can't go into hole</span></div>
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<span style="background-color:rgb(255,255,255);display:inline" class="">Best regards,</span></div>
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<span style="background-color:rgb(255,255,255);display:inline" class="">Shasha</span></div>
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<span id="cid:183c3cfb9ffd2a6827e1"><Screen Shot 2022-10-10 at 1.31.48 PM.png></span><br class="">
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