Re: [ccp4bb] Micromatrix seeding using the mosquito

2010-09-29 Thread Jürgen Bosch 
Not with membrane proteins, however we use our Mosquito with 20 nl seeding 
standard.

Strip 1 Protein, Strip 2 Seed stock solution
We first copy the plate by either picking up protein, then reservoir and 
dispensing together 200-400 nl or we multidispense protein and add the 
reservoir later (larger drops, in particular if we can't concentrate the 
protein well then we add 600 nl and hope that enough evaporates before we come 
with the reservoir solution).
As last step we add the 20 nl seed solution.
We've also done seed titrations then we use three strips 1)Protein 2) seed 
buffer solution 3) seed. In that case the combination of 2+3 is always 100nl 
and we start out with 5 nl Seed stock.
In the IntelliPlates (3well) we play the protein/reservoir ratio game + seed
Reproducibility is pretty good. We keep our seed stock where we grew the 
crystals e.g. 20˚C or 4˚C.

Jürgen
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Sep 29, 2010, at 5:23 AM, Patrick Shaw Stewart wrote:

> Hi Jeroen
>  
> I think that part of the point of the method is that it’s incredibly quick 
> and easy to do – you can make seeds and run an experiment in 20 mins.  But 
> I’m sure it’s important to use the seed stock quickly and freeze it.
>  
>  
> Maria
>  
> I think most people use 100 nl seed stock, 200 nl reservoir, 300 nl protein 
> as D’Arcy and co originally suggested.  Maybe more seed stock would be better 
> as Roberto says, to increase the additive effect.  The main reasons not to 
> put seed stock into the reservoir are (1) it’s more work – compared to your 
> robot picking up the seed stock from a PCR tube - and (2) sometimes your seed 
> stock is very valuable, for example if you only have one crystal and you make 
> up say 5 ul of seed stock.
>  
> Has anyone had success with MMS microseeding with membrane proteins?  I 
> wonder if membrane protein crystals dissolve more easily.
>  
> Patrick
>  
>  
>  
> --
> For information and discussion about protein crystallization and automation, 
> please join
> our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en
>  
>  patr...@douglas.co.ukDouglas Instruments Ltd.
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>  Directors: Peter Baldock, Patrick Shaw Stewart
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>  
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of mesters
> Sent: 28 September 2010 07:26
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Micromatrix seeding using the mosquito
>  
> Hello Maria,
> 
> the success of this method depends on a few things.
> First, your seed stock-solution. Most people prepare it by smashing up seeds 
> in mother liquor (i.e. reservoir solution). Looking at the phase diagram, you 
> will quickly understand that this might not be the best way to go as you will 
> dissolve your seeds fairly quickly over time. That is why people prepare 
> really fresh stocks, keep them on ice and freeze them as soon as possible in 
> order to "stabilize" the seeds. Keeping on ice will only help if your protein 
> is less soluble at lower temperatures, which true for roughly 45% of proteins 
> (45% will react retrograde, 10% do not show a clear preference). Looking at 
> the phase diagram again, you will also understand that seeding only works for 
> "near" equilibrated drops that are metastable-supersaturated. If 
> undersaturated, seeding will not work as the seeds will dissolve over time.
> 
> Keeping the above considerations in mind, best is to seed after the drops 
> have equilibrated. Next best option is to add the seeds or the protein as the 
> last component to the drop...
> 
> Jeroen.
> 
> 
> 
> 
> On 20.09.10 09:53, Maria Håkansson wrote:
> Dear all,
>  
> Anyone who have had success using the mosquito for micromatrix seeding?
> Which way is the optimal way of adding seed solution to sitting drops on an 
> MRC plate?
>  
> Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir 
> solution or
> to add concentrated seed solution (1 microliter) directly to the reservoir 
> solution and then pipett
> 100 nl protein + 100 nl reservoir solution.
>  
> Best regards and thanks in advance,
>  
> Maria Håkansson
>  
>  
> 
> __
> Maria Håkansson, Ph.D.
> Senior Scientist, Max-lab, Lund University

Re: [ccp4bb] Micromatrix seeding using the mosquito

2010-09-20 Thread Alexey Rak 
dear Maria,
we routinely use Mosquito for MMS, the ratio works the best: 100nl protein + 
multi-aspirate mode x (15nl seeds + 85nl reservoir solution).
best regards,
alexey
 
Alexey RAK

Structural Biology

SDI/LGCR

Sanofi-aventis R&D

Centre de Recherche Paris

13, Quai Jules Guesde - BP 14

94403 Vitry sur Seine Cedex

France

 

Phone : +33 (0)1 58 93 86 93

Fax : +33 (0)1 58 93 80 63

Mobile : +33 (0)6 26 71 28 60

Email : alexey@sanofi-aventis.com

 
 
 
 
 
 
 
 
 
 
 


From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Steiner, 
Roberto
Sent: Monday, September 20, 2010 10:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Micromatrix seeding using the mosquito


Dear Maria

In our hands Mosquito MMS worked by adding 2x protein solution + 1x seed 
solution + 1x reservoir. The reason for doing this was two-fold:

I) ratio protein/reservoir is similar to the condition that generated the seed 
stock  
II) a deliberate bias toward the initial condition is given


I) and II) might not always be helpful and possibly other ratios should be 
explored.

Best wishes
Roberto


On 20 Sep 2010, at 08:55, "Maria Håkansson"  
wrote:



Dear all, 

Anyone who have had success using the mosquito for micromatrix seeding?
Which way is the optimal way of adding seed solution to sitting drops 
on an MRC plate?

Is it better to add 100 nl protein + 10 nl seed solution + 100 nl 
reservoir solution or
to add concentrated seed solution (1 microliter) directly to the 
reservoir solution and then pipett
100 nl protein + 100 nl reservoir solution.

Best regards and thanks in advance,

Maria Håkansson






__
Maria Håkansson, Ph.D.
Senior Scientist, Max-lab, Lund University

Phone: +46 (0) 76 8585 706
Fax: +46 (0) 46 222 47 10
Ole Römers väg 1 (P.O. Box 188)  
SE-221 00 Lund, Sweden   


Web address:  <http://www.maxlab.lu.se> www.maxlab.lu.se  

Email:  <mailto:maria.hakans...@maxlab.lu.se> 
maria.hakans...@maxlab.lu.se
__





<>

Re: [ccp4bb] Micromatrix seeding using the mosquito

2010-09-20 Thread Steiner, Roberto 
Dear Maria

In our hands Mosquito MMS worked by adding 2x protein solution + 1x seed 
solution + 1x reservoir. The reason for doing this was two-fold:

I) ratio protein/reservoir is similar to the condition that generated the seed 
stock
II) a deliberate bias toward the initial condition is given

I) and II) might not always be helpful and possibly other ratios should be 
explored.

Best wishes
Roberto

On 20 Sep 2010, at 08:55, "Maria Håkansson" 
mailto:maria.hakans...@maxlab.lu.se>> wrote:

Dear all,

Anyone who have had success using the mosquito for micromatrix seeding?
Which way is the optimal way of adding seed solution to sitting drops on an MRC 
plate?

Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir 
solution or
to add concentrated seed solution (1 microliter) directly to the reservoir 
solution and then pipett
100 nl protein + 100 nl reservoir solution.

Best regards and thanks in advance,

Maria Håkansson



__
Maria Håkansson, Ph.D.
Senior Scientist, Max-lab, Lund University
Phone: +46 (0) 76 8585 706
Fax: +46 (0) 46 222 47 10
Ole Römers väg 1 (P.O. Box 188)
SE-221 00 Lund, Sweden

Web address:  www.maxlab.lu.se
Email:  
maria.hakans...@maxlab.lu.se
__

[ccp4bb] Micromatrix seeding using the mosquito

2010-09-20 Thread Maria Håkansson 

Dear all,

Anyone who have had success using the mosquito for micromatrix seeding?
Which way is the optimal way of adding seed solution to sitting drops  
on an MRC plate?


Is it better to add 100 nl protein + 10 nl seed solution + 100 nl  
reservoir solution or
to add concentrated seed solution (1 microliter) directly to the  
reservoir solution and then pipett

100 nl protein + 100 nl reservoir solution.

Best regards and thanks in advance,

Maria Håkansson




__
Maria Håkansson, Ph.D.
Senior Scientist, Max-lab, Lund University
Phone: +46 (0) 76 8585 706
Fax: +46 (0) 46 222 47 10
Ole Römers väg 1 (P.O. Box 188)
SE-221 00 Lund, Sweden

Web address: www.maxlab.lu.se
Email: maria.hakans...@maxlab.lu.se
__