James,
I think you need to be a little more specific about what you want to
calculate. Keq for a reaction A <=> B will not change with an enzyme
mutation as the thermodynamic relationships between the reactants and
products do not change. As a catalyst, the enzyme impacts only the
free
As Artem and Ezra have already mentioned the basic aspects of
isolating IBs, I should point out that most people wash the pellet of
the first low-speed centrifugation 2-3 more times. While Ezra
suggests using increasing amounts of urea, you may have to tailor the
composition of the wash bu
Sajid,
It is not clear what you are asking. Are you asking whether there are
common structural features in domains that bind nucleotide di/tri
phosphates and dinucleotides? Or are there clear structural rules to
consider when designing a (di)nucleotide binding site. If it is the
former,
Marek,
Your observation is not unusual. We have seen it for cases with 2
independent molecules in the ASU (Mulichak et al. Proc. Natl. Acad.
Sci. USA 100, 9238-43, 2003.) and a "symmetric" 222 tetramer in the
ASU (Mulichak et al., Biochemistry 41, 15578-89, 2002). Continue
refinement, a
James,
Dima is right. One of the protocols for protein-refolding with
sarcosyl uses cyclodextrins to remove it. However, sarcosyl is an
anionic detergent above pH 5.5 under most conditions, so any ion
exchange platform should work well to markedly reduce its presence in
a preparation.
Parveen,
Bert and Pascal are correct in that most alkyl glycoside detergent are
notoriously difficult to crystallize in aqueous solution when you have
the beta-anomer (what we normally buy). However, the alpha-anomers
can be quite easy to crystallize and can contaminate batches of beta-
a
Jacob,
Although the use of patch-clamp pipettes and micromanipulators come to
mind, I just wonder about what would happen to the pipette and the
fluid in it when you flash-froze it. Nonetheless, the idea of a means
to transfer small crystals easily and quickly has some merit. In
another
Look into the 0.5 sq. m Millipore Pellicon ultrafiltration/
microfiltration devices (http://www.millipore.com/catalogue/module/
c558). They are moderately expensive, but the membranes are robust,
reusable and allow fast concentration steps. We have regularly used
them to concentrate cells
Jacob,
DDM should never crystallize under any aqueous conditions seen for
protein crystallization. We tend to store 20% stock solutions (~0.4
M) of DDM in buffer at 4C. DDM tends to phase out long before it
would crystallize (~ 0.8 mol percent).
With this said, how have you set up the s
To all,
For those of us who are do-it-yourselfers and like to work at home
(and who may have tons of tape), here is something of interest:
http://arstechnica.com/journals/science.ars/2008/10/22/home-made-x-
rays-and-other-fun-with-tape
I suppose we could dope the tape with different metals
Meg,
I might add that if you have boiled your samples, you might disregard
this:
1) Instead of contamination, you might just be seeing multiple
bands due to 'aggregation' of your protein! Make sure you boil the
sample prior to loading on gel and also that your loading dye
contains SDS,
Bill,
We have used the texts “The Organic Chemistry of Enzyme-Catalyzed
Reactions” (revised edition) by Silverman (2002) and Alan Fersht's
book "Structure and Mechanism in Protein Science: A guide to Enzyme
Catalysis and Protein Folding (1999).
Michael
**
Mark,
A little more information on the protein and need would be nice. Is
it a large peptide, a small protein, or a recombinant protein? Do
you want real quantitative results or semi quantitative (like BCA,
Bradford, or Lowry which can be off by 20% or more relative to the
[BSA])? Do y
I would not call that publication as the final arbiter either, but it
is close. My wife, who teaches acid-base chemistry to literally 1500
students a year, rolled her eyes when she heard about this discussion
and said "Didn't you guys know that?" She pointed me to a slightly
more definiti
Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: [EMAIL PROTECTED]
R.M. Garavito wrote:
Buffer making is very muc
Buffer making is very much an empirical process, but there is a
comment that needs to be made about the use of the H-H equation and
pKa values. I have to teach our department's biochemistry
laboratory, and I sadly would have to take off points from all the
discussions as the H-H equation
Subbu,
Take a look at the following for an example of how to proceed.
J. Ay et al., Proc. Natl. Acad. Sci. USAVol. 95, pp. 6613–6618, June
1998 Structure and function of the Bacillus hybrid enzyme GluXyn-1:
Native-like jellyroll fold preserved after insertion of autonomous
globular domain
Evette,
A good machine shop can make a 1 L Amicon-like tirred-cell
concentrator quite easily (and many have). But another alternative
is the Pellicon Tangential Flow Filtration Cassettes (see http://
www.millipore.com/techpublications/tech1/pb022). The Pellicon XL 50
cassettes (50 sq. cm
Matthew,
You're not going to ruin your column, but you won't get great
performance either. Elution by pH change is a very common method,
but getting a really linear pH gradient is very hard. The Mono Q
matrix is a strong anion exchanger, meaning that it is insensitive to
pH changes, i.e
The primary reason they don't have much on the Sephadex resins is
that the are quite old-fashioned (and also relatively cheaper than
most of their high-end resins). However, old Pharmacia/LKB had some
really great manuals on chromatography that are still quite useful
for new biochemists.
cryogenic gas which I
believe is why CF4 is not used as much.
For more tips on cryo-cooling, see also the PDF linked at http://
www.rigaku.com/cryo/ and the references therein.
Jim
On Fri, 6 Jun 2008, R.M. Garavito wrote:
Tommi,
The question has been asked and answered not by protein
c
Tommi,
The question has been asked and answered not by protein
crystallography, but by cyroelectron microscopy and EM freeze etch
research. Even as far back as the early 1960's, people noticed that
liq. N2 was really slow at cooling. Read the cyroEM work on the
bacteriorhodopsin photocyc
Weikai,
You might look up these references:
A. Lustig et al. "Density determination by analytical
ultracentrifugation in a rapid dynamical gradient: application to
lipid and detergent aggregates containing proteins." Biochim.
Biophys. Acta 1115 (1991) 85-95.
Ariel Lustig et al. "Molecula
Jacob,
I doubt that the spherulites formed in the presence of detergents are
"primary" detergent phenomenon. However, detergent micelles and the
protein-detergent aggregates (please don't say protein-detergent
micelles) will condense (phase) out in the presence of high salt or
polymers t
Alfredo,
It's not clear from your email what your purpose is: to illuminate a
crystal/sample on a microscope stage or on a goniostat. In either
case, you might check out Ocean Optics (http://
www.oceanoptics.com/). They have based their systems on fiber optic
light sources and CCD detec
Calcium citrate does have a relatively low solubility (~15 mM in the
cold), and its solubility decreases as the temperature goes up.
Thus, getting calcium citrate crystals is a possibility if both are
at 200 mM. However, you can get sodium citrate up to ~1.4 M at
least, depending on the p
DDM does not form crystals in aqueous solution. The published phase
diagrams show this. In fact, few of the alkyl glycoside detergents
crystallize if the sugar group is a beta anomer, even in organic
solvents. This is one of the reasons why they are so difficult to
purify by recrystalliz
Kay,
Go with LiCl as a reservoir solution (not necessarily in the protein
drop). It can generate a very low water vapor pressure. It will
suck the water out of almost anything. The CRC handbook has all the
information although buried deep within it. However, the DNA and
polysacchari
Melody,
While Joe addressed the possible effects of detergents on the
crystallization of proteins, the possible effects (both negative and
positive) of detergents on proteins are quite variable. Often there
are no general rules except to avoid ionic detergents (from anionic
detergents l
Nian,
It is also important to point out that gluteraldehyde is quite
penetrating (which is why it is used as a fixative in EM) and
volitile. A 1% solution is 100 mM, which is quite concentrated.
Adding gluteraldehyde by vapor diffusion is quite effective and
gentle, but does take a bit
Donghui,
We have several membrane proteins crystallized in salt-based
conditions, actually in high sodium citrate. Yes, LiCl or lithium
formate are good alternatives, but also try lithium citrate, as
citrate also depresses the freezing point quite well.
Good luck,
Michael
*
Jacob,
This is not a uncommon problem with Ni-chelation chromatography (on
Ni-NTA or Talon) with soluble and membrane proteins. In most of my
experiences, it is a salt problem (i.e., too little), but
hydrophobicity issues abound, as well.
On a pilot scale, just add any of the suggested a
Joe,
As David said many different types of salt crystals can grow in the
presence of phosphate, even when the concentrations of PO4 or many
divalent cations are VERY low (10s of micromolar or less). Struvite
is common (NH4MgPO4) when ammonium sulfate is the precipitant, mM
magnesium is
Chen and David,
Before adding detergent, be forewarned that the MPB in many fusions
will not bind to an amylose column in the presence of most
detergents, particularly maltoside detergents. It has been the bane
to us so we have engineered MBP vectors with His tags to deal with
this. Wha
Kay,
I beg to differ, but only in a pedantic way. Historically, Rsym
would refer to the agreement in symmetry-related reflections within a
single data set and Rmerge would be the agreement between 2 or more
data sets that were merged. This was the way we did it back in the
"old day" of
Joyce,
In the US, we have often purchased HA compounds from Strem (http://
www.strem.com). They make a lot of different organometallic
compounds as well.
Michael
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecula
Jacob,
Savvas' suggestion about the the Calbiochem and Anatrace product
catalogs is a good one, particularly as the latter is fairly up to
date regarding the newest detergents. Reviews tend to be out of date
quickly.
Also, concerning methods and the odd useful measurements of detergent-
Joe,
What your are describing is microcrystal formation. The silkiness or
"opalescence" is very typical of microcrystalline showers.
Concerning the effect of DTT, I recall one other case where DTT-
sensitive microcrystals formed: the crystalline insecticidal toxins
produced by B. thuring
After Bill Scott's and Juergen Bosch's comments about upgrading to
Leopard 10.5, it has been quiet. As I am upgrading some "non-
essential" machines up to 10.5, I just wanted to check if there is
more info out there.
Despite horror stories on the discussion site, upgrading 1 G4 PB, an
Int
Martin,
Yes, I forgot the "bible" of detergents: "Critical micelle
concentrations of aqueous surfactant systems" by Mukerjee and
Mysels. However, the problem with all of these surfactant sources,
including the Surfactant science series mentioned by Sue Roberts, is
that they are focused
Jacob,
Actually, there is no real definitive compendium of detergents and
their properties (solubility, CMC, aggregation number, etc.).
Because of their importance to industry, commercial compendia, as
Anatrace's catalog, are often the most complete. However, most
commercial compendia a
Saavas and Tommi,
The questions of what is the detergent content of a membrane protein
crystal and how to explicitly determine the amount of detergent in a
crystal are extremely difficult to address. Moreover, is it
worthwhile to even attempt to correct the Matthews coefficient? I
perso
Simon,
This is not an unusual situation with sulfate, and, yes, sulfate
often occupies phosphate binding sites (particularly when you're work
with 1-3 molar concentration of ammonium sulfate). The simplest way
to remove sulfate from the crystal is to transfer to high
concentrations of ci
Douglas and Ronaldo,
I wanted to put in my two cents worth on both of your queries at the
same time. You should look up glycerol dehydrogenase from the yeast
S. pombe. Sp-GlyDH was solved accidently a few years ago by Anne
Mulichak in our group (PDB 1TA9). We were trying to crystallize
Hubing,
Your problem may actually be a detergent concentration mismatch
between your mother liquor and the cryosolution. This particularly
happens with vapor diffusion setups: there is a delicate balance of
"free" detergent in the mother liquor versus the proportion of the
detergent wh
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