Re: [ccp4bb] crystal with precipitation

- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: crystal with precipitation
From: shivesh kumar 2shivesh {- at -} GMAIL {- dot -} COM
Date: 2007-08-22

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From: Jan Abendroth jan {- dot -} abendroth {- at -} GMAIL {- dot -} COM
Date: 2007-08-22

Subject: Re: crystal with precipitation
From: Lisa A Nagy lisanagy {- at -} UAB {- dot -} EDU
Date: 2007-08-22

Oh, there is do much you can do!
Lots of alternatives:
1. Decrease or increase the protein concentration (with a corresponding
increase or decrease of precipitant.
2. Use a temperature gradient.
3. Set up in a capillary with liquid-liquid diffusion
4. SEED!

#3 or #4 are your best bets, probably.

For the capillaries, using Hamilton syringes:
Pipet 20 ul protein in a 50 ul capillary. Ease it away from the end.
When you are done, make sure you can reach the liquid with the syringe
from the other side.
Hold your finger on one end. Seal the other end with sticky wax
Layer 5 ul water on top.
Carefully layer the precipitant (10-30 ul) on top of the water.
You might want to use a 4mM glass capillary mounted on a syringe,
instead of Hamiltons.
Seal the other end.
Wait 3 days.
Then look.

Naturally, you can scale this down using 20 ul capillaries, or even
smaller X ray capillaries.
But then you can't use syringes to load them, use smaller capillaries.
On the upside, you could shoot directly from the experiment.

This has worked well for me.

Lisa

-----Original Message-----
From: CCP4 bulletin board on behalf of shivesh kumar
Sent: Wed 8/22/2007 3:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystal with precipitation

Dear all
I am trying to crystallize a 7kDa protein using MPD as a precipitant at
16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is
that the drop is precipitating in one day only and the crystals are
coming
with precipitation.I have added 5% glycerol and 100mM of Nacl as an
additive
with the mother liquor to avoid precipitation.But,the precipitation is
still
there.The volume of the mother liquor is 500 microlt.The drop size is
2+2.I
welcome all the suggestions regarding avoiding the precipitation.Thanx
is
advance.
Shivesh kumar

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: crystal with precipitation
From: shivesh kumar 2shivesh {- at -} GMAIL {- dot -} COM
Date: 2007-08-22

Next message:
Subject: Re: mosflm and APS BM17
From: Jan Abendroth jan {- dot -} abendroth {- at -} GMAIL {- dot -} COM
Date: 2007-08-22