[3dem] How to improve particle distribution on GO-coated ultrAufoil

[Buerger]

Hi,
I would try Quantifoil carbon grids R2/4+ or R3/3+ 2nm additional carbon film. Check allways NS first and then go into cryo. Use 100%humidity and 4C. Blotting only 2 or 4 seconds. If you succeed then you increase the sample concentration by the factor of 10 and freeze 1 or 2sec R1.2/1.3 without carbon. Good luck. I know every grid looks different. Best Jörg.

Am 30. März 2020 05:15:26 MESZ schrieb Satoru Machida <dbssator at nus.edu.sg>:
>Dear all
>
>I prepared cryo grids for single particle analysis for 600 kD
>protein-RNA complex. The particles are overcrowded. After dilution, the
>particles are not separated. When I diluted it further, holes became
>empty. Gel filtration makes a single peak, but on the grid they stick
>together. I already tried glycerol, DMSO, amphipols, salt concentration
>70-400 mM. None of them worked well. The buffer contains 150 mM NaCl,
>10 mM HEPES pH8, 0.5 mM EDTA, 4 mM DTT. The sample had about 1mg/ml,
>blotted for 3 sec , force 0, 100% humidity, 22C on Ultraufoil R0.6/1.0
>coated with graphene-oxide. The image was taken by Tecnai12. The
>particles are hard to get into the holes without graphene-oxide.
>Any expert suggestions will be helpful.
>
>Kind regards
>Machida
>
>________________________________
>
>Important: This email is confidential and may be privileged. If you are
>not the intended recipient, please delete it and notify us immediately;
>you should not copy or use it for any purpose, nor disclose its
>contents to any other person. Thank you.

-- 
Diese Nachricht wurde von meinem Android-Gerät mit K-9 Mail gesendet.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20200330/b8d84590/attachment-0001.html>