Hi there, all good suggestions to optimize crystals, here are few other things
to try:
1. Spin down your protein sample in a centrifuge prior to setting up trays to
remove protein aggregates.2. Incubate trays at lower temperatures (for example
if crystals grow at RT, try 15 and 4 C).3. Try streak seeding (Hampton Research
sells a nice seeding tool and provides a protocol).4. Try using an additive
screen (also available from Hampton Research).
Hope it helps,John
On Sunday, December 8, 2024 at 11:41:33 AM CST, Patrick Shaw Stewart
<patr...@douglas.co.uk> wrote:
It is pretty normal (actually, pretty good) to get crystals within 3 days under
room temperature
Why work with conditions that take 3 days for crystals to grow when you can,
very likely, find conditions that grow in one day by using seeding ! ?
(I'm simplifying slightly - they may take 3 days to stop growing)
On Sat, Dec 7, 2024 at 8:00 PM Kevin Jin <kevin...@gmail.com> wrote:
First of all, you need to verify these crystals are protein. To do so, use a
loop to fish some crystals and take a shot to see the reflection pattern, a
lazy way!
If they are protein, then start considering the optimization. No matter which
kind of kits you have used, they are just matrices of different conditions.
It is pretty normal (actually, pretty good) to get crystals within 3 days under
room temperature. The only problem is the xtals grow a little bit fast,
Notice your condition, protein solubility is not an issue. The only problem is
the folding force needed to be adjusted. Likely, you need to decrease the total
salt concentration.
For PEG 8K, you may try 5 ~ 15%, instead of 20~25 %.
Different polymers, their pH values are different. Based on your protein's
charge, different combinations may give you some unexpected results, dynamics
vs. kinetics process.
Basically, inorganic chemistry + polymer chemistry/physics + solution chemistry
+ protein chemistry
Good luck with best regards!
On Thu, Dec 5, 2024 at 6:18 PM 白雪慧 <zb20193020...@cau.edu.cn> wrote:
Thank you very much for your reply to my question on how to optimize
microcrystal. In response to your suggestions and questions, I would like to
make the following reply:
First of all, my protein can grow crystals like this one in a variety of
situations.
For example, other conditions:
1.15% PEG 8000;0.2M MgCl2;0.1M HEPES pH 7.5
2.20% PEG 1500;0.2M MgCl2;0.1M HEPES pH 7.5
3.25% PEG 8000;0.2M NaCl;0.1M NaCaco pH 5.5
4.20% PEG 1e500;0.2M MgCl2;0.1M Tris pH 8.5
Second, my crystals grow within three days, and there is no phosphate in the
protein solution.
Finally, in fact, I tried crystal inoculation before, but did not find any
change in crystal shape. Maybe my operation was also wrong. I sucked out the
crystal, mashed it, diluted it in series, and removed the primary crystal
condition, and the protein concentration did not change, nor did the
concentration of the pool liquid change greatly
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
--
patr...@douglas.co.uk Douglas Instruments Ltd.
Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek
http://www.douglas.co.uk
Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034
Regd. England 2177994, VAT Reg. GB 480 7371 36
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
########################################################################
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list
hosted by www.jiscmail.ac.uk, terms & conditions are available at
https://www.jiscmail.ac.uk/policyandsecurity/
