January 2020 3:52 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation
trials
Hi Armando,
I echo Rodger’s advice at 10 mg/ml to start. Our high-throughput
crystallization lab has a FAQ page that notes this
https://hwi.buffalo.edu/high-throughput
board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger
Rowlett
Sent: Wednesday, January 8, 2020 11:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation
trials
I usually set a partial screen, maybe 24-48 conditions. If less than half the
Dear Armando,
in case you have enough material to spare, I would use a concentrator to
create a saturated solution, i.e. concentrate until it precipitates. Measure
the concentration of the saturated solution, i.e., supernatant without
disturbing the solution with the precipitate.
You can start
I usually set a partial screen, maybe 24-48 conditions. If less than half
the wells have precipitate, double protein concentration. If most have
precipitate, maybe reduce protein or halve concentration of screen
reagents. I usually start at 10 mg/mL or so. You can conveniently change
protein conc.
Dear all,
I was wondering how to guess the optimal protein concentration for the initial
crystallisation trials. Is there any trick or assay other than the classic PCT
from Hampton?
Armando
To unsubscribe from the CCP4BB
If using Unicorn,
1)Open your chromatogram in Evaluation window.
2)Go to Operations-> Pool
3)Choose which baseline estimation suits you, define the pools (numbered rulers
that appear under the chromatogram)
4)type in the path length (2 or 10 mm for UV-900 and 2 mm for UPC-900)
5)type in the mass
CP4BB@JISCMAIL.AC.UK
Sent: Wednesday, 15 January 2014 11:09 PM
Subject: [ccp4bb] Protein concentration form chromatograms
Dear all,
A question for the biochemistry-inclined folks in the bb; how do I calculate
protein concentration of chromatography fractions, starting from Abs280 from
the UV mo
Thanks for all the replies. And I would not call this laziness, rather
crowdsourcing
I should have added a few more details; it is easy to export from say
Unicorn to excel, a list of pairs of values, Abs280 vs elution times, with
which one can recreate the chromatogram. I wanted to use these f
I am not sure, but probably path length not 1 cm in a detector cuvette .
You must refer to your HPLS system manual for this value
15.01.2014 19:09, Karel Chaz пишет:
Dear all,
A question for the biochemistry-inclined folks in the bb; how do I
calculate protein concentration of chromatography f
On 1/15/14, 9:34 AM, Matthew Franklin wrote:
- I usually approximate the peak as a triangle, so that the total peak
area is 1/2 height (absorbance maximum) x base (the number of ml in
your pool)
GE's UNICORN for AKTAs (and probably the new Biorad FPLC software)
integrates and tells you the are
Hi Karel -
To add to what Jurgen said, a few points on the measurement of the
protein peak from the chromatogram.
- I usually approximate the peak as a triangle, so that the total peak
area is 1/2 height (absorbance maximum) x base (the number of ml in your
pool)
- If your peak is a strong
Step1:
visit Protparam tool and C&P your sequence, scroll down until you find the
extinction coefficient part. If OD280 is not close to 1 then make sure to take
that into account in your chromatogram say it is 0.7
step2: look at your mAU's
If your peak is 1000 mAU then you have 0.7 mg/ml in that
Dear all,
A question for the biochemistry-inclined folks in the bb; how do I
calculate protein concentration of chromatography fractions, starting from
Abs280 from the UV monitor? I know I could figure it out myself if I really
tried, but why bother when I have access to so many brilliant minds...
an spending 2 ul on prescreening.
>
> ** **
>
> Just an opinion sans statistics, BR
>
> ** **
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
> *Debasish
> Chattopadhyay
> *Sent:* Tuesday, June 11, 2013 1:02 AM
> *To:* CCP4BB@JI
Tuesday, June 11, 2013 1:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein concentration for crystallization
Perhaps my question was not expressed well. I wanted to know if proteins
crystallize more frequently when the protein concentration is in the range
5-30mg/ml.
The a
.UK>
Subject: [ccp4bb] Protein concentration for crystallization
What would be a convenient way to estimate what percentages of proteins have
been crystallized in a concentration range, for example 5-30 mg?
Debasish Chattopadhyay
University of Alabama at Birmingham
CBSE-250
1025 18th Street
Best Regards
Rana
From: Debasish Chattopadhyay
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, June 10, 2013 4:49 PM
Subject: [ccp4bb] Protein concentration for crystallization
What would be a convenient way to estimate what percentages of proteins have
been crystallized in a concentration range, for
Dear Debasish,
you can use REMARK 200 field in pdb file. Sadly, this field is not
mandatory so not everyone provide protein concentration info.
10.06.2013 18:49, Debasish Chattopadhyay ?:
What would be a convenient way to estimate what percentages of
proteins have been crystallized in a
What would be a convenient way to estimate what percentages of proteins have
been crystallized in a concentration range, for example 5-30 mg?
Debasish Chattopadhyay
University of Alabama at Birmingham
CBSE-250
1025 18th Street South, Birmingham, Al-35294
USA
Ph: (205)934-0124; Fax: (205)934-0480
to:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger
Rowlett
Sent: Thursday, July 19, 2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein concentration vs Molecular wt...
This is almost exactly our basic approach, too. Before we got a dropsetter, we
did 24 wells (1/2 screen) to get a fee
See http://www.douglas.co.uk/PDB_data.htm, section 4
On 19 July 2012 20:58, james09 pruza wrote:
> Dear Crystallographers,
> Is there any rule of thumb for Protein concentration and molecular weight
> for crystallization trials of a soluble protein? Looking for high molecular
> wt. protein ~50
Hi James,
You can get the PCT (Pre-Crystallisation Test) from one of the more
famous manufacturers of crystallography products - essentially you can
quickly screen your protein to get an idea if it is too dilute, too
concentrated, or somewhere in the middle. Of course, the results are
representati
l Message-
From: james09 pruza
To: CCP4BB
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular
weight for crystallization trials of a soluble protein? Looking for
ther 1 mg/ml or 10mg/ml or 100mg/ml is reasonable.
Mark
-Original Message-
From: james09 pruza
To: CCP4BB
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molec
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular weight
for crystallization trials of a soluble protein? Looking for high molecular
wt. protein ~50kDa.
James.
Hello Teresa,
c = x/(N.V)
where x=number of molecules per unit cell, N=Avogadro's number, V=volume of
the unit cell in liter, c=concentration in molar
Cheers
Filip
On Mon, Dec 13, 2010 at 12:07 PM, Teresa De la Mora wrote:
> Hi all
>
> I'm looking for a way to calculate the protein concent
If you know the Matthews volume, then you can convert to protein
concentration with:
[protein] (mg/mL) = 1/( V_M (A^3/Da) ) * 1.66e-21 (mg/Da) * 1e24 (A^3/mL)
For example, lysozyme crystals (V_M = 2.0 A^3/Da) contain about 830
mg/ml of protein. Most protein crystals are in this ballpark (be
@JISCMAIL.AC.UK
Subject: [ccp4bb] protein concentration in crystal
Hi all
I'm looking for a way to calculate the protein concentration in a single
crystal. So what I'm thinking is to use the Matthew's number to calculate
how many molecules inside the crystal, then multiply
Sounds like a good units conversion problem for
Introductory Chemistry. Basically, you can get molecules per cubic
angstrom from your unit cell analysis (molecules per unit cell and
cubic angstroms per unit cell) and convert to the units of your
choice, mg/cm^3,
Hi all
I'm looking for a way to calculate the protein concentration in a
single crystal. So what I'm thinking is to use the Matthew's number to
calculate how many molecules inside the crystal, then multiply that
number by Avogadro's number to get moles then divide by volume of
crystal. Is
Mark,
There are two very easy alternatives. First is the Bradford Coomassie Dye
binding assay. You can buy a kit from Biorad at the following link:
http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=0349900713.1219329576&BV_EngineID=ccchadeemgdfflfcfngcfkmdhkkdfll.0&c
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
Often the exact concentration is not so important compared to the
ability to establish proportional read-out, ease and reproducibility
so that systematic variations and comparisons can be made. For
considerations of molar ratios of for example protein:ligand complexes
one would often screen
Dear Mark
Most easy way to have relative estimation is to run PAGE of your protein
with known proteins of known different concentration so u can have a good
relative estimation.
but for more accurate estimation u can go for Biochemical methods such as
Lowry method or BCA method which are more se
Dear all,
I would be glad to hear what (simple) method I should use to determine
protein concentrations as accurately as possible. Presently, I'm
measuring absorption at 280nm with a nanodrop device. I either have 0
or 1 tryptophan and no activity test.
Many thanks in advance!
Best regar
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