Dear Prashant,
I have been working with a protein-protein complex expressed in mammalian
cells, and that complex in very poorly soluble. Even with 500mM NaCl in the
buffer, I cannot concentrate the complex to above 3 mg/mL. I tried an old
school technique and precipitated my protein complex
Some memories from the dark recesses of my mind… Hope it helps!
Bryan
From: Prince, D Bryan
Sent: Thursday, August 14, 2014 6:07 PM
To: 'Gloria Borgstahl'
Subject: RE: [ccp4bb] dynapro DLS cuvettes
Dear Gloria,
I seem to remember that we had a DynaPro DLS system and used disposable plastic
One thing you can try is to place modeling clay at the base of the pin. Add
some mother liquor to a quartz capillary (1-2mm should work) and under a
microscope carefully cover the loop with the capillary, pressing it into the
clay to seal the crystal in a closed system. As long as you move or
Sorry, forgot you didn’t have any quartz capillaries. If you are in a biochem
lab, do you have access to hematocrit capillaries? You might be able to use
them to cover the loop in the fashion I posted just a minute ago. Again, good
luck!
Bryan
Dear Jacob,
Please check the following references:
http://www.ncbi.nlm.nih.gov/pubmed/8771063
http://www.ncbi.nlm.nih.gov/pubmed/9251095
From some limited reading of another paper, it seems like the dark solution
could be the conversion of L-Dopa into melanin, which is accelerated in
Of course, if you can plan ahead, you can grow your crystals in shipping
friendly plates. See the In-Situ plate at Mitegen for details.
http://www.mitegen.com/mic_catalog.php?c=insituplates
you also might try to add agarose to the drop to fix the crystal in place, but
that could be tough with a
I recently had a similar situation for a protein crystal that was sitting in
very thick, sticky goop. I put three drops of Paratone N on a coverslip and
harvested the crystal by using a Mitegen yoke tool to dig around the crystal,
then a regular nylon loop to move the crystal into a drop of
At AstraZeneca, we're constantly striving to make new discoveries.
Discoveries that will make a meaningful difference to health globally.
We already have an exceptional product pipeline, and everyone here has a
part to play in making sure that pipeline fulfills its potential. This
is your
Are you referring to the I3C magic phasing triangle by any chance? Beck,
et al Acta Cryst D 61(?) (2008) is the reference I think.
Good luck!
Bryan
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Richard Gillilan
Sent: Friday, November 30, 2012 5:21 PM
To:
Those are the EasyXtal 15 well tools with the EasyXtal DG-Crystal Supports.
Look at this link to find them:
http://www.qiagen.com/products/protein/crystallization/default.aspx
I agree that they keep the drops from spreading out, but I have experienced
trouble harvesting smaller crystals from
Dear Muhammed,
In my experience, crystals like that are likely made up of contaminated or
non-homogeneous protein. Have you run NATIVE PAGE and IEF gels to determine the
purity of your sample? Is it the correct MW by mass spec without contaminating
peaks?
Good Luck!
Bryan
From: CCP4
Ed,
I looked into this a number of years ago, and remember the following papers. I
did not actually use any of the databases described due to IT issues at the
time. I hope this helps.
Kind regards,
Bryan
LISA: an intranet-based flexible database for protein crystallography project
management
Good afternoon fellow ccp4bb'rs,
I was wondering if anyone knows if a still to condense gaseous propane
to liquid propane using dry ice is commercially available. I want to
make sure that it is not something I can purchase before I build one fit
to purpose. I appreciate any advice and knowledge
I thought we had evidence for hackers doing this already. J
http://www.nature.com/nature/journal/v477/n7365/full/477373e.html
(no flames, please-'tis intended to be funny, not factual)
Bryan
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Jacob Keller
Sent:
Actually, I would refer the ccp4-bbs to Journal of Structural Biology
175 (2011) pp216-223 for the use of fluorescence in relation to protein
crystallization.
Regards,
Bryan
--
Confidentiality Notice: This message is
Dear Theresa,
Gary Gilliland's paper on cryosalts would seem to be useful for your problem.
http://scripts.iucr.org/cgi-bin/paper?en0028
Also, I have used 15% glycerol in a synthetic mother liquor to effectively
freeze crystals grown in 2M Ammonium Sulfate. Another method that Jim Pflugrath
I remember a technique that used warmed Vaseline (liquefied) in a shallow pan.
People would invert the plate and dip it into the liquefied Vaseline, then flip
it over to dry. I suppose one could use masking tape to cover the outer edge of
the plate to keep it Vaseline free. I am not sure how it
I thought I would have nothing to say about this topic, but I am sitting
here at my desk listing to my iPod, remembering fondly my apple IIc
computer I started with in elementary school. I would like to share a
story with you, if you would be kind enough to listen. I have a son who
is severely
Dear Flip,
I think with respect to the Formulatrix database, it would be useful to
have the date of entry into the database for each screen input. I agree
that there are discrepancies in the database, but they can generally be
traced to a change from one catalog to the next. If you have the date
I had a similar problem with crystals that were obtained from PEG-based
precipitants over long periods of time. If I harvested the crystals in
about 5 days, they would diffract to ~3 Angstrom. If I let them grow any
time past about 7 days, they became PEG-alated and didn't diffract at
all. I
Dear TY,
Typically between 5-10x molar concentration over the protein is enough to
ensure binding when the IC50 is uM to low mM. For tighter binding compounds (nM
to low uM), 2-5x is sufficient. Whatever you do, when the precipitate occurs DO
NOT REMOVE it. I learned to my chagrin that you
Something else to try would be the Protic Ionic Liquid kit from Hampton.
I recently had crystals of a protein that would only grow as laminated
stacks of plates. Optimizing the conditions and using an additive screen
didn't improve crystal morphology. I tried the PIL kit from Hampton and
was able
For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark.
Dear Arnon,
I have a Nanodrop2000, which reads from the post or a user supplied
cuvette. I have had NO complaints about using the Nanodrop for reading
protein concentration immediately prior to crystallization setup. When I
have observed differences in OD280 vs Bradford, it is usually due to one
Hello all:
I am curious about what level of recovery is reasonable when performing
a TEV cleavage to remove 6HIS tags (N-terminal) from a protein.
Currently we are experiencing 50% loss of soluble protein after TEV
cleavage, and feel that this is too much. Are there better systems for
his tag
Dear Harvey,
Microseeding and adding 1-5% glycerol to the drops helped me when I
obtained crystals like these. Good Luck!
Bryan
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Harvey Rodriguez
Sent: Sunday, February 20, 2011 9:29 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject:
Hello Steve,
You can also check out this paper: Bystrom, Pettigrew, Remington and
Branchaud (1997) Bioorganic Medicinal Chemistry Letters, Vol 7 No 20
pp2613-2616. It describes the creation of AMPPCF2P, which I had
opportunity to use a few years back and it worked great!
Good luck,
Bryan
From:
I think that this CRO can help you:
Proteos
4717 Campus Drive
Kalamazoo, Michigan 49008
269.372.3423
http://www.proteos.net
Good luck!
Bryan
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Junyu Xiao
Sent: Tuesday, February 08, 2011 2:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Dear Chandan,
388 buffers are quite a bit. I presume you are talking about intervals of pH
and not 388 buffers all at one pH. Can you explain a bit more about how you
determined that the buffer is the problem, and what results led you to that
conclusion?
From: CCP4 bulletin board
Quickly passing the crystal through Paratone N has worked well for me
when I crystallize in ammonium sulfate or sodium citrate conditions.
Another trick is to dissolve sucrose (table sugar) in 10uL of the
reservoir solution until it is saturated. Then separate the
sucrose-reservoir mix into two
I recently ran mass spec analysis on some crystals that I had obtained
from an optimization screen. I was looking for modifications in the
protein. In order to get enough signal, I had to harvest and dissolve
about 8 crystals roughly 0.3 x 0.15 x 0.15mm into the MS loading buffer
in order to get a
Dear Xuan,
I am not certain, but I think that Jacob was referring to a
spectrophotometer called a Nanodrop. It is available from ThermoFisher
Scientific and can provide absorbance data on as little as 2uL of
sample. I think that if you have access to a Nanodrop, and use Ultrafree
0.5mL
Having recently completed the CSHL Macromolecular crystallography course, I can
recommend Introduction to Macromolecular Crystallography by Alexander McPherson
(ISBN 987-0-470-18590-2). I am posting the link below:
My most sincere and heartfelt condolences to Elspeth and her family. May
they find peace of mind and strength of heart during this most difficult
time.
Kind regards,
Bryan Prince
On Jul 3, 2010, at 6:28 AM, Frank von Delft wrote:
Dear all
Last night, John Barnett, physicist and husband of
If you check
http://formulatrix.com/product_crystallizationimaging_muvis.html, you
will find a stand-alone UV microscope. I have enjoyed using the
RockImager UV microscope, which is an integrated hotel/imaging station.
The MUVIS is a bench-top standalone imaging system that should supply
what you
When the ACA meeting was in Hawaii (2006?), there was information about
microwaving PEG solutions to artificially age new samples so that they would
crystallize like the older PEG's. So I would infer that heat does have a
significant effect on solid PEG's. All PEG's with MW =600 are solids at
Dear Jerry,
First of all, it will be hard to reproduce the conditions with the
glycosylated protein because by its nature, it is heterogeneous. One
thing I would try with the glycosylated protein is a detergent screen,
or if you don't have one, use a few NDSB's. Second, I would try setting
up
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