Hi Keith,
PEG3350 freezes ~30% in combination with 10% glycerol, since you
already have some glycerol from your protein I would increase both PEG
and glycerol say 30% PEG3350 and 20% glycerol - but you tried this
already.
Your real problem is the big crystal, you might want to consider
breaking it into two or three pieces and then freeze the smaller
chips. Alternatively you can harvest your crystals before they get
that big or add something that prevents them from growing preferably
into one direction (if you have the HR additive screen try it in
combination with your condition).
Another option is have you tried to flash anneal your crystal wet and
dry ? That could have very surprising results.
What type of loops do you use to mount ? As large as the crystal ?
Then you have lots of liquid which needs to be frozen as well and then
the amount of glycerol will not be sufficient. How do you freeze LN2
or in the stream (hopefully blocked stream prior to freezing) ?
Just some thoughts. Good luck,
Jürgen
On 14 Feb 2009, at 21:15, Keith Romano wrote:
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Biochemistry and Molecular Biology, W8708
615 North Wolfe Street
Baltimore, MD 21205
Phone: +1-410-614-4742
Fax: +1-410-955-3655
