From: anita p
Date: 9 September 2011 12:22
Dear Crystallographers,
I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it.
This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition.
Could any one suggest a remedy for this.
with regards
Anita
----------
From: Poul Nissen
Yes - add glycerol to the reservoir -
15% glycerol has a huge influence on the vapour diffusion gradients
Poul
----------
From: David Roberts
The following is simply a quote from a recent post by Enrico Stura. Probably has a lot to do with what you are finding:
"Glycerol is also great to reduce nucleation. If you decide to add glycerol to the protein solution (for solubility, but in your case it might be for stability
reasons), you also need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and
not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m "
Good luck
Dave (but really, this is all Enrico)
----------
From: George T. DeTitta
Dear Anita
You are seeing the effects of a very modest reduction of vapor pressure of water with polymers. You could add salt to your reservoir and you could insure your drops are at the same temperature as your reservoir - not easy in conventional "constant temp" incubators.
George
Sent via BlackBerry by AT&T
----------
From: Enrico Stura
Anita,
see the message I posted yesterday on the CCP4bb:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg22638.html
or google "stura glycerol"
Re: [ccp4bb] How to help crystal grow bigger
In your case the sentence you need is:
"You need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context: Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762. " Enrico.
--
----------
From: anita p
Thanks a lot for your suggestions.
Would it be worth while to try and remove glycerol at the last step which is gel filtration and then set trays in my case?
I have never tried without glycerol.
reg.
Anita
----------
From: Ed Pozharski
Sure, assuming that your protein does not become unstable without
glycerol (protein solubility is likely to decrease too). On a general
note, protein crystallization is the (dark) art of trying as many things
as you possibly can (including the crazy stuff). Quoting the great
Bruce Foxman, you need to use the crystallization method number eight,
which is the one that works :)
--
Hurry up, before we all come back to our senses!
Julian, King of Lemurs
Date: 9 September 2011 12:22
Dear Crystallographers,
I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it.
This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition.
Could any one suggest a remedy for this.
with regards
Anita
----------
From: Poul Nissen
Yes - add glycerol to the reservoir -
15% glycerol has a huge influence on the vapour diffusion gradients
Poul
----------
From: David Roberts
The following is simply a quote from a recent post by Enrico Stura. Probably has a lot to do with what you are finding:
"Glycerol is also great to reduce nucleation. If you decide to add glycerol to the protein solution (for solubility, but in your case it might be for stability
reasons), you also need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and
not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m "
Good luck
Dave (but really, this is all Enrico)
----------
From: George T. DeTitta
Dear Anita
You are seeing the effects of a very modest reduction of vapor pressure of water with polymers. You could add salt to your reservoir and you could insure your drops are at the same temperature as your reservoir - not easy in conventional "constant temp" incubators.
George
Sent via BlackBerry by AT&T
----------
From: Enrico Stura
Anita,
see the message I posted yesterday on the CCP4bb:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg22638.html
or google "stura glycerol"
Re: [ccp4bb] How to help crystal grow bigger
In your case the sentence you need is:
"You need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context: Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762. " Enrico.
----------
From: anita p
Thanks a lot for your suggestions.
Would it be worth while to try and remove glycerol at the last step which is gel filtration and then set trays in my case?
I have never tried without glycerol.
reg.
Anita
----------
From: Ed Pozharski
Sure, assuming that your protein does not become unstable without
glycerol (protein solubility is likely to decrease too). On a general
note, protein crystallization is the (dark) art of trying as many things
as you possibly can (including the crazy stuff). Quoting the great
Bruce Foxman, you need to use the crystallization method number eight,
which is the one that works :)
--
Hurry up, before we all come back to our senses!
Julian, King of Lemurs
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