<html>
<head>
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
<style type="text/css" style="display:none;"> P {margin-top:0;margin-bottom:0;} </style>
</head>
<body dir="ltr">
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
Hi 3DEMers,</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
My lab had previous success with 2nm continuous carbon coated Quantifoils (QF R2/2 300 +2nm, or EMS cat # Q3100CR2-2NM), but hydration and particle adsorption stopped working when we got two new boxes (both are lot 201027). I tested the chloroform, acetone,
isopropanol cleaning procedure from Passmore and Russo 2016, checked by negative stain and a very stable control protein (confirmed to work on a CF400-UL grid with the same glow discharge and stain procedure). On the QF+2nm with or without cleaning, particles
aggregate in pooled stain patches (images attached). The failure in cryo-EM is similar, very dry squares, sometimes with central pools, but hardly any particles.<br>
</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
<br>
</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
Has anyone seen this behavior with continuous carbon grids, especially the commercial coatings? Any recommendations for other cleaning techniques?</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
<br>
</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
Unfortunately the glow discharge treatment has to be quite gentle otherwise the thin coating over the holes will break - we use 15 mA for 15 sec and then try to compensate with longer droplet incubation times, 20-60 seconds.</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
<br>
</div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
Thanks for any advice!<br>
</div>
<div>
<div class="elementToProof" style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
<br>
</div>
<div id="Signature">
<div>
<div style="font-family:Arial,Helvetica,sans-serif; font-size:12pt; color:rgb(0,0,0)">
</div>
<div id="divtagdefaultwrapper" dir="ltr" style="font-size:12pt; color:#000000; font-family:Arial,Helvetica,sans-serif">
<div name="divtagdefaultwrapper" style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:; margin:0">
<font size="2"><span style="font-family:Arial,Helvetica,sans-serif">--</span></font></div>
<div name="divtagdefaultwrapper" style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:; margin:0">
<span style="font-family:Arial,Helvetica,sans-serif"></span><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Dan Richman, PhD</span><span id="ms-rterangepaste-start"></span><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">
</span><font size="2"><span style="font-size:8pt; font-family:Arial,Helvetica,sans-serif">(</span><font size="2"><span style="font-size:8pt; font-family:Arial,Helvetica,sans-serif">he/him/his</span></font><span style="font-size:8pt; font-family:Arial,Helvetica,sans-serif">)</span></font></font><span id="ms-rterangepaste-end"></span></font>
<div><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Research Associate, Berger Lab</span></font></div>
<div><font size="2"></font><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Dept of Biophysics and Biophysical Chemistry</span></font></div>
<div><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Johns Hopkins University School of Medicine</span></font>
<div><span style="font-family:Arial,Helvetica,sans-serif"></span>
<div>
<div><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">mobile: 201-669-0967</span><font size="2"></font></font></div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</body>
</html>