Re: [ccp4bb] How to get rid of Membrane formed on hanging droplets?

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CCP4bb <-- 2007 <-- March 2007 <-- 07 March 2007

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Subject: Re: How to get rid of Membrane formed on hanging droplets?
From: Frank von Delft frank {- dot -} vondelft {- at -} SGC {- dot -} OX {- dot -} AC {- dot -} UK
Date: 2007-03-07

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Subject: Re: XDS to MTZ for SOLVE
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-03-07

Subject: Re: How to get rid of Membrane formed on hanging droplets?
From: Maneesh Yadav yadavm {- at -} SCRIPPS {- dot -} EDU
Date: 2007-03-07

Something that has worked for me (most of my crystals have been a little chunkier, but it's helped even with
delicate small needles as well): just add about pipette a microliter of reservoir solution on top of your drop. I find this tends to dissolve smaller xtals, and I think some combination of turbulence and dilution dislodges bigger ones from membranes and plastic/glass. I even do this with 100 nanoliter drops (i.e. add a microliter on top), you may want to do it a 4 deg. to slow dilution as much as possible.

-----Original Message-----
From: CCP4 bulletin board on behalf of David Briggs
Sent: Mon 3/5/2007 7:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] How to get rid of Membrane formed on hanging droplets?

Hi,

What you describe is "skin" - rumoured to be denatured protein.
http://xray.bmc.uu.se/~terese/crystallization/pics/SKIN2.JPE

And it's a pain, especially when your crystals stick to it.

Best suggestions are:

1) Try screening additives or detergents that might stabilise it
2) Try changing the crystallisation setup to microbatch under oil - I have
never had skin that way.

If you can't get rid of the skin, you're going to have to excise your
crystals out very carefully.
This has worked for me in the past:

1) Put a big blob (~10-20ul) of 50:50 Paratone oil : Mineral Oil on top of
the drop.
This does two things - prevents the drop drying out as your wrestle away the
skin and can be your cryoprotectant as well.

2) Use a fine accupuncture needle or hampton probe to cut the patch of skin
away with your crystal attached.

3) manoeuvre the crystal+skin away from the drop (but still in the oil) and
then very very very carefully start trying to pull the skin away with the
probe or a loop (or even both - one in each hand)

4) The Oil will give you plenty of time to play with your crystal to try and
get the skin off. By the time you've done this you will also have got most
excess mother liquor away so you can go right ahead and freeze your crystal.

This requires patience, but the more skin you get away the better the freeze
and the less you will harm your diffraction.

It goes without saying that if you can get a big xtal NOT attached to the
skin, then life is much easier.

Good Luck!

Dave

On 05/03/07, Wang, Yeming (NIH/NIEHS) [F] wrote:
>
> Dear everyone,
>
> I am working on crystallization of a protein/RNA complex recently. The
> crystals were initially grown from BICINE( 9.0) 0.1M, 1,4-Dioxane 2%(v/v)
> , PEG 20,000 10%(w/v), at 10mg/ml. I noticed that there was a membrane
> formed on the surface of the hanging droplets. This membrane seems very
> sticky. Consequently, almost all of the crystals stick to this membrane and
> can't be seperated for data collection. Sitting drops were also tried but
> crystals stick to the bottom of the sitting well. Different buffer(Tris,
> CHES), different PEG(PEG 8000, PEG3350, from 10%-1%) and different protein
> concentration (10-3mg/ml) were also tried, but the sticky membrane was still
> there. Does anyone have some experience solving this problem? Any
> suggestions would be highly appreciated!
>
> Yeming
> ---------------------
> Yeming Wang, Ph.D.
> Laboratory of Structural Biology: Macromolecular Structure Group
> National Institute of Environmental Health Sciences
> National Institute of Health
> Mailing Address: Street Address:
> NIEHS, MD F3-05 NIEHS, Building 101, Room F363
> P.O. BOX 12233 111 T.W. Alexander Drive
> RTP, NC 27709 RTP, NC 27709
> Tel (o): 919-316-4634
> E-mail: wangy2@niehs.nih.gov
>

--
---------------------------------------
David Briggs, PhD.
Father & Crystallographer
www.dbriggs.talktalk.net
iChat AIM ID: DBassophile
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Next message:
Subject: Re: XDS to MTZ for SOLVE
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-03-07