[ccp4bb]: Summary of oil and cryo-protectant combo.

["Tom Caradoc-Davies" <toms_postingacct@hotmail.com>]

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Hi All,
       as per normal here is a summary of the emails etc about the use of 
oil and cryo-protectant together:

The technique described was placing a layer of paraffin oil over a drop 
containing xtals in cryo-solution so that once the xtal is looped out 
through the oil a thin oil film holds the xtal in the loop and prevents 
dehydration. This film provides at least 10 minutes for the xtal to be 
placed in the cryo-stream. The use of oil also produces a smaller volume of 
cryo-protectant, as this clings to the faces of the xtal leaving the edges 
poking into the oil layer, and a resulting smaller diffuse solvent ring.

1. It seems that while the use of oil for mounting xtals is common, labs 
tend to use either oil OR a cryoprotectant for a particular xtal. If people 
do combine the two together as I described above this method does not seem 
to be commonly used (unless I misunderstood Stephan Ginell's Email!). :)

2. Martyn Symmons has a nice trick of making a skirt out of a cryo-vial and 
sealing this with grease to make a "well" around a drop on a coverslip which 
can be filled with oil. This prevents the drop from drying out allows more 
time to loop out the xtal.

3. Kris Tesh, Peter Moody, Harry Powell, Stephan Ginell and Henry Bellamy 
let me know that oil is used in many labs for several steps in xtal growth, 
mounting and by itself as a cryo-protectant.

4. Several of the above also pointed out that some components of the mother 
liquor such as isopropanol may enter the oil phase. This was anticipated by 
Hope in the original papers describing the use of oil and Henry wrote "... 
things like isopropanol can partition into the oil.  I may be wrong but I 
think that Hope would saturate the Paratone with the mother liquor by 
shaking them together and then letting the phases separate."

5. Henry Bellamy also pointed out that "You need to be very careful about 
immersion oil, it is selected for refractive index and can contain all kinds 
of stuff.  Back in the 80's Ivan Rayment was sealing Linbro plates with 
immersion oil and he could only get crystals after he switched to another 
sealant.  So if things don't work the immersion oil is a good first choice 
for something to change." If anyone would like to try the 
oil/cryo-protectant technique I would recommend using  paraffin oil as this 
avoids the above problem, is cheap and worked the best out of a range of 
oils I tested including the 4 Hampton immersion oils and ParatoneN. I used 
LABCHEM brand bulk paraffin by AJAX chemicals but any lab-grade paraffin 
should do.

6. Peter Moody had experienced problems when using a 50:50 Paratone/paraffin 
mix with the oil being used as the sole cryo-protectant. The oil mix took up 
some of the aqueous phase during xtal mounting and become cloudy when 
cryo-cooled to 100K.

7. Ho-Leung Ng pointed out that a convenient bath of liquid N2 can save 
rushing to the cryo-stream. However this still requires the use of 
cryo-transfer and the oil/cryo combo is much quicker when screening a lot of 
xtals and does not have the risk of damaging a xtal via momentary warming.

Thanks to everyone for the input and advice and if anyone else would like 
more information please send me an Email and I will send a detailed 
protocol.
Cheers,
        Tom

	Tom Caradoc-Davies
	Biochem Dept
	Univ. Otago
	New Zealand

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