Re: [ccp4bb] Off-topic question related to ITC binding studies

[ABHISHEK SUMAN]

Hi Gergő,

Thank you for discussing it in detail. It is a great help. Based on the
curve fit, we also believe that the two proteins bind independently to the
DNA duplex with the same affinity. The DNA indeed contains 2 sites for
protein binding. As far as the dimerization of protein is concerned, we
have already confirmed, through different approaches, that the protein
doesn't behave as a dimer in the solution.

Regards
Abhishek


*Dr. Abhishek Suman*

*Ph.D (Structural Biology)*

Indian Institute of Technology Hyderabad

Kandi 502 284 Sangareddy

Telangana INDIA

Contact: +91 91002 74548, +91 80843 11898

Email: abhisheks.i...@gmail.com



P* Please don't print this e-mail or attachment unless necessary. Preserve
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On Wed, Aug 17, 2022 at 12:24 AM Gergő Gógl  wrote:

> Dear Abhishek,
>
> When you use a function with "one set of sites" and an “independent”
> model, you most likely used some sort of quadratic binding formula. In case
> the true binding equation is like you wrote [2A + B <-> (A2)B], then it
> is not expected to look like a quadratic, but rather like a cubic formula.
>
> {Just solve this: Kd=( ( Atot - 0.5*[(A2)B] )^2 * (Btot - [(A2)B]) ) / ( [
> (A2)B] ) and you will see that the resulting function is cubic for the
> concentration of the complex ([(A2)B]).}
>
> If you indeed had a perfect sigmoidal observation with no systematic
> divergence from the fitted quadratic curve, I would not find any strong
> indication that the binding function should be considered as cubic.
> Instead, I would assume that:
>
> -the protein binding to the DNA follows a simple bimolecular binding model
> and the DNA just happens to be able to bind two molecules of proteins
> independently with the same affinity (within the detection thresholds of
> the assay). [A + B_site1 <-> AB_site1 and A + B_site2 <-> AB_site2]
> -or the protein binds as a stable dimer to the DNA molecule and therefore
> the binding can be considered as pseudo bimolecular. (DNA+dimer <->complex)
> In this case, you should change the protein concentration to "protein dimer
> concentration" during fitting. [(A2) + B <-> (A2)B]
>
> In either case, your dissociation constant has a dimension of M and not
> M^2.
>
> If your binding model really follows the reaction you proposed and you do
> not like the first option where the affinities of the sites are identical,
> you should use a "two independent set of sites" model. In this case, you
> will end up with 2 independent affinities that are not identical and their
> dimension will be in M. (If they are close, you are better if you use a one
> site and assume that the sites are not distinguishable.)
>
> If your sites are not independent and you have strong cooperativity, you
> will need to use more complex biochemistry (such as sequential binding) and
> your Kd dimension may be different, but this is most likely not the case
> because you had a decent fit with a simple.
>
> I hope I could help and did not miss something important. Please let me
> know if others propose something different!
>
> Best,
> Gergo
>
> ABHISHEK SUMAN <2fccd9428006-dmarc-requ...@jiscmail.ac.uk> ezt írta
> (időpont: 2022. aug. 16., K, 19:40):
>
>> Hello everyone!
>>
>>
>>
>> Hope this email finds you well. I have an off-topic question regarding
>> ITC binding studies, which was asked by a reviewer.
>>
>>
>>
>> We performed an ITC binding study (using Affinity ITC, TA Instruments) to
>> evaluate protein-DNA interaction which resulted in a perfect sigmoidal
>> curve. We used the ‘one set of sites’ binding algorithm (“independent”
>> model) for curve fitting and to calculate binding and thermodynamic
>> parameters. The study suggested two copies of the protein binding to a
>> single duplex DNA, i.e., the stoichiometry of protein:DNA is 2:1 (N=2). The
>> ITC calculated the KD (equilibrium molar dissociation constant) in μM
>> (micromolar). But the reviewer is asking to report the KD in
>> (micromolar)^2 instead of micromolar mentioning that the binding reaction
>> is 2A + B <-> (A2)B and the complex is (A2)B and not AB. Though we're
>> trying to explain to the reviewer that we couldn't find any software that
>> can compute the KD in (micromolar)^2 for the stoichiometry of 2 but he
>> is not agreeing to it. We have used the NanoAnalyze software from the TA
>> instrument. This software does not have a model to measure the KD in
>> (micromolar)^2.
>>
>>
>> I would be grateful if you could help me to resolve this problem or at
>> least let me know what explanation might be appropriate to answer the
>> reviewer’s concern that it’s a general practice to report the KD in the
>> Molar irrespective of stoichiometry.
>>
>>
>>
>> Thanks in advance.
>>
>>
>>
>> Regards
>>
>> Abhishek
>>
>>
>> *Dr. Abhishek Suman*
>>
>> *Ph.D (Structural Biology)*
>>
>> Indian Institute of Technology Hyderabad
>>
>> Kandi 502 284 Sangareddy
>>
>> Telangana INDIA
>>
>> Contact: +91 

[ccp4bb] RES: [ccp4bb] Off-topic question related to ITC binding studies

[Rafael Marques]

I am not a proper chemist, but I remember that during my college classes my 
professor emphasized that ^x is a measured value and not really related to 
stoichiometry. If someone has something to add…

Best

Rafael Marques da Silva
Mestre em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"


De: ABHISHEK SUMAN<mailto:2fccd9428006-dmarc-requ...@jiscmail.ac.uk>
Enviado:terça-feira, 16 de agosto de 2022 14:41
Para: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Assunto: [ccp4bb] Off-topic question related to ITC binding studies

Hello everyone!

Hope this email finds you well. I have an off-topic question regarding ITC 
binding studies, which was asked by a reviewer.

We performed an ITC binding study (using Affinity ITC, TA Instruments) to 
evaluate protein-DNA interaction which resulted in a perfect sigmoidal curve. 
We used the ‘one set of sites’ binding algorithm (“independent” model) for 
curve fitting and to calculate binding and thermodynamic parameters. The study 
suggested two copies of the protein binding to a single duplex DNA, i.e., the 
stoichiometry of protein:DNA is 2:1 (N=2). The ITC calculated the KD 
(equilibrium molar dissociation constant) in μM (micromolar). But the reviewer 
is asking to report the KD in (micromolar)^2 instead of micromolar mentioning 
that the binding reaction is 2A + B <-> (A2)B and the complex is (A2)B and not 
AB. Though we're trying to explain to the reviewer that we couldn't find any 
software that can compute the KD in (micromolar)^2 for the stoichiometry of 2 
but he is not agreeing to it. We have used the NanoAnalyze software from the TA 
instrument. This software does not have a model to measure the KD in 
(micromolar)^2.

I would be grateful if you could help me to resolve this problem or at least 
let me know what explanation might be appropriate to answer the reviewer’s 
concern that it’s a general practice to report the KD in the Molar irrespective 
of stoichiometry.

Thanks in advance.

Regards
Abhishek

Dr. Abhishek Suman
Ph.D (Structural Biology)
Indian Institute of Technology Hyderabad
Kandi 502 284 Sangareddy
Telangana INDIA
Contact: +91 91002 74548, +91 80843 11898
Email: abhisheks.i...@gmail.com<mailto:abhisheks.i...@gmail.com>

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[ccp4bb] Off-topic question related to ITC binding studies

[ABHISHEK SUMAN]

Hello everyone!



Hope this email finds you well. I have an off-topic question regarding ITC
binding studies, which was asked by a reviewer.



We performed an ITC binding study (using Affinity ITC, TA Instruments) to
evaluate protein-DNA interaction which resulted in a perfect sigmoidal
curve. We used the ‘one set of sites’ binding algorithm (“independent”
model) for curve fitting and to calculate binding and thermodynamic
parameters. The study suggested two copies of the protein binding to a
single duplex DNA, i.e., the stoichiometry of protein:DNA is 2:1 (N=2). The
ITC calculated the KD (equilibrium molar dissociation constant) in μM
(micromolar). But the reviewer is asking to report the KD in (micromolar)^2
instead of micromolar mentioning that the binding reaction is 2A + B <->
(A2)B and the complex is (A2)B and not AB. Though we're trying to explain
to the reviewer that we couldn't find any software that can compute the KD
in (micromolar)^2 for the stoichiometry of 2 but he is not agreeing to it.
We have used the NanoAnalyze software from the TA instrument. This software
does not have a model to measure the KD in (micromolar)^2.


I would be grateful if you could help me to resolve this problem or at
least let me know what explanation might be appropriate to answer the
reviewer’s concern that it’s a general practice to report the KD in the
Molar irrespective of stoichiometry.



Thanks in advance.



Regards

Abhishek


*Dr. Abhishek Suman*

*Ph.D (Structural Biology)*

Indian Institute of Technology Hyderabad

Kandi 502 284 Sangareddy

Telangana INDIA

Contact: +91 91002 74548, +91 80843 11898

Email: abhisheks.i...@gmail.com



P* Please don't print this e-mail or attachment unless necessary. Preserve
trees on the planet.*

-- 


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Re: [ccp4bb] OFF TOPIC question

[Peat, Tom (Manufacturing, Parkville)]

Hello Anamika,

>From the information you gave, you have two different promoters- araC and the 
>lac promoter. LacI is the lac inhibitor. The lac promoter is a two part 
>system- when lactose is not present, the inhibitor sits near the promoter and 
>blocks transcription of genes downstream.
Almost any E. coli system will work the expression of proteins from these 
promoters, excepting those that have been modified in some way to block 
arabinose or lactose use. The BL21 (DE3) system has the T7 system implemented, 
but as you are not using the T7 promoter, there is no particular reason to use 
this version of E. coli.
Best of luck, tom

Tom Peat, PhD
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board  on behalf of Anamika Singh 

Sent: Thursday, November 26, 2020 8:59 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] OFF TOPIC question

Hi all,

I have two constructs having different ori, p15ori and M13 ori, different 
promoters araC and LacI, and different antibiotic resistance chloramphenicol 
and Ampicillin respectively. I would like to know which expressing E. coli host 
cells will be good for the co-transformation of these constructs?

Thanks

Anamika




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Re: [ccp4bb] OFF TOPIC question

[Crissy L Tarver]

BL21(DE3) pLysS using 5X KCM and heat shock transformation worked for me.

Crissy L Tarver
Postdoctoral Researcher
Department of Structural Biology
Stanford University School of Medicine

From: CCP4 bulletin board  on behalf of Hughes, Jonathan 

Sent: Thursday, November 26, 2020 2:48:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] AW: [ccp4bb] OFF TOPIC question


Bl21Pro

j



Von: CCP4 bulletin board  Im Auftrag von Anamika Singh
Gesendet: Donnerstag, 26. November 2020 11:07
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] OFF TOPIC question



One correction to the previous question: I have two constructs having different 
ori, p15ori and M13 ori, different promoters araBAD promoter and LacI, and 
different antibiotic resistance chloramphenicol and Ampicillin respectively. I 
would like to know which E. coli host cells will be good for the 
co-transformation of these constructs?



On Thu, 26 Nov 2020 at 11:59, Anamika Singh 
mailto:anamika.ii...@gmail.com>> wrote:

Hi all,



I have two constructs having different ori, p15ori and M13 ori, different 
promoters araC and LacI, and different antibiotic resistance chloramphenicol 
and Ampicillin respectively. I would like to know which expressing E. coli host 
cells will be good for the co-transformation of these constructs?



Thanks



Anamika






--

Dr. Anamika Singh
Post-Doctoral Fellow

Silberman Institute of Life Sciences

Hebrew University of Jerusalem, Israel

No: 054-294-8036





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[ccp4bb] AW: [ccp4bb] OFF TOPIC question

[Hughes, Jonathan]

Bl21Pro
j

Von: CCP4 bulletin board  Im Auftrag von Anamika Singh
Gesendet: Donnerstag, 26. November 2020 11:07
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] OFF TOPIC question

One correction to the previous question: I have two constructs having different 
ori, p15ori and M13 ori, different promoters araBAD promoter and LacI, and 
different antibiotic resistance chloramphenicol and Ampicillin respectively. I 
would like to know which E. coli host cells will be good for the 
co-transformation of these constructs?

On Thu, 26 Nov 2020 at 11:59, Anamika Singh 
mailto:anamika.ii...@gmail.com>> wrote:
Hi all,

I have two constructs having different ori, p15ori and M13 ori, different 
promoters araC and LacI, and different antibiotic resistance chloramphenicol 
and Ampicillin respectively. I would like to know which expressing E. coli host 
cells will be good for the co-transformation of these constructs?

Thanks

Anamika



--
Dr. Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036



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Re: [ccp4bb] OFF TOPIC question

[Anamika Singh]

*One correction to the previous question:* I have two constructs having
different ori, p15ori and M13 ori, different promoters *araBAD promoter*
and LacI, and different antibiotic resistance chloramphenicol and
Ampicillin respectively. I would like to know which E. coli host cells will
be good for the co-transformation of these constructs?

On Thu, 26 Nov 2020 at 11:59, Anamika Singh  wrote:

> Hi all,
>
> I have two constructs having different ori, p15ori and M13 ori, different
> promoters araC and LacI, and different antibiotic resistance
> chloramphenicol and Ampicillin respectively. I would like to know which 
> *expressing
> E. coli host cells* will be good for the co-transformation of these
> constructs?
>
> Thanks
>
> Anamika
>
>

-- 
Dr. Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036



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[ccp4bb] OFF TOPIC question

[Anamika Singh]

Hi all,

I have two constructs having different ori, p15ori and M13 ori, different
promoters araC and LacI, and different antibiotic resistance
chloramphenicol and Ampicillin respectively. I would like to know
which *expressing
E. coli host cells* will be good for the co-transformation of these
constructs?

Thanks

Anamika



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Re: [ccp4bb] Off-topic question

[Jan Stransky]

Hi,
there is a guide here, but you should get the proper script for Pymol
from the Consurf server.
http://www.protein.osaka-u.ac.jp/rcsfp/supracryst/suzuki/jpxtal/Katsutani/en/consurf.php
Jan

On 6/22/19 10:24 PM, khaja faisal tarique wrote:
> Hi everyone,
> 
> I was wondering can anyone suggest me how to project the primary
> sequence conservation calculated through the Consurf server.  Any help
> or suggestion will be appreciated. I have pasted a figure from an
> article just for reference.
> 
> 
> Best
> 
> Khaja
> 
> image.png
> 
> 
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 



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[ccp4bb] Off-topic question

[khaja faisal tarique]

Hi everyone,

I was wondering can anyone suggest me how to project the primary sequence
conservation calculated through the Consurf server.  Any help or suggestion
will be appreciated. I have pasted a figure from an article just
for reference.


Best

Khaja

[image: image.png]



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[ccp4bb] OFF topic Question Nucleotide exchange of RAS protein?

[Anamika Singh]

Hi All,

Does anyone know what is the actual difference between CIP and EDTA method
for exchanging the GppNHp (non-hydrolysable GTP) or GDP with the purified
RAS protein?

I have a protocol according to which I need to incubate the purified RAS
with GppNHp having 20U of alkaline phosphatase, 10uM of Zinc Chloride and
200mM ammonium sulfate for 2 hr at room temperature. In this protocol, they
have mentioned that ammonium sulfate needs to be added in the last. I am
quite confused, according to them do I need to add it after 2 hrs
incubation or I have to add this at last to the mixture of (purified
RAS+GppNHp+Zinc chloride and alkaline phosphatase)?

Since this protein and its mechanism is new to me so not sure what to do.
After reading some papers also I am not able to understand which method is
good for exchange and how these reactions behave?

Please help me in this regard.

-- 
Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036



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Re: [ccp4bb] Off topic question

[Georg Hochberg]

Dear Reza,

CD hit will do exactly that.

Cheers,
Georg

Sent from my iPhone

On Jan 3, 2019, at 2:41 PM, Reza Khayat 
mailto:rkha...@ccny.cuny.edu>> wrote:


​Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] Off topic question

[Javier Gonzalez]

Hi Reza, happy new year!
The choice would depend on your alignment (aminoacid or nucleotides? are
the sequences closely or distantly related? is it a large alignment? are
there many gaps?)... Anyway, I think the safest, unbiased way to determine
a group of outliers might be to compute a phylogenetic tree and look for an
outgroup. But if the set of sequences is too large you might want (first)
to use a clustering algorithm, such as CD-HIT (
http://weizhongli-lab.org/cd-hit/).
HTH,
Javier

On Thu, Jan 3, 2019 at 6:08 PM Ethan A Merritt 
wrote:

> On Thursday, January 3, 2019 12:40:05 PM PST Reza Khayat wrote:
> > ?Hi,
> >
> >
> > Happy new year to all!  A bit of an off topic question.  Does anyone
> know of a method/program to extract the most distinct "n" (n>2) sequences
> from a sequence alignment?  Thanks.
>
> If these putative "most distinct" sequences are hypothesized to belong
> together,  then i suggest K-means clustering.  If they are hypothesized
> to be unrelated individual outliers then I think you would just take the
> worst scores using whatever metric you used to create original alignment.
>
> Ethan
>
> >
> >
> > Best wishes,
> > Reza
> >
> >
> > Reza Khayat, PhD
> > Assistant Professor
> > City College of New York
> > Department of Chemistry
> > New York, NY 10031
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> MS 357742,   University of Washington, Seattle 98195-7742
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>


-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  LinkedIn




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Re: [ccp4bb] Off topic question

[Ethan A Merritt]

On Thursday, January 3, 2019 12:40:05 PM PST Reza Khayat wrote:
> ?Hi,
> 
> 
> Happy new year to all!  A bit of an off topic question.  Does anyone know of 
> a method/program to extract the most distinct "n" (n>2) sequences from a 
> sequence alignment?  Thanks.

If these putative "most distinct" sequences are hypothesized to belong
together,  then i suggest K-means clustering.  If they are hypothesized
to be unrelated individual outliers then I think you would just take the
worst scores using whatever metric you used to create original alignment.

Ethan

> 
> 
> Best wishes,
> Reza
> 
> 
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742



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Re: [ccp4bb] Off topic question

[Zhijie Li]

PCA?

On Jan 3, 2019, at 3:41 PM, Reza Khayat 
mailto:rkha...@ccny.cuny.edu>> wrote:


​Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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[ccp4bb] Off topic question

[Reza Khayat]

?Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] Off-topic question

[Debanu Das]

Hi,
I was going to suggest the same but since it has already been said, here’s a 
cheeky suggestion: you could try determining the crystal structure of the 
complex and get a direct sequence readout for the nuclei acid.
Best,
Debanu 

> On Mar 5, 2018, at 9:34 PM, Philippe BENAS 
> <0d88e888355a-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi Jobi,
> 
> Phenol/CHCl3 extraction, iPrOH precipitation and then nucleic acid sequencing.
> 
> Best regards,
> Philippe
>  
> Philippe BENAS, Ph.D.
> 
> Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
> Faculté de Pharmacie, Université Paris Descartes
> Case 48
> Av, de l'Observatoire
> F-75270 PARIS cedex 06
> +33.1.5373.1599
> E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr
> URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , 
> http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18
> 
> 
> 
> De : Jobichen Chacko <jobich...@gmail.com>
> À : CCP4BB@JISCMAIL.AC.UK 
> Envoyé le : Mardi 6 mars 2018 5h11
> Objet : [ccp4bb] Off-topic question
> 
> Dear All,
> We are trying to identify/sequence a DNA/RNA fragment (around 100bp) which 
> was co-purified along with our protein.
> The expression was done in E.coli.
> Any suggestions on how to do this.
> Thank you.
> Jobi
> 
> 

Re: [ccp4bb] Off-topic question

[Philippe BENAS]

Hi Jobi,
Phenol/CHCl3 extraction, iPrOH precipitation and then nucleic acid sequencing.

Best regards,Philippe Philippe BENAS, Ph.D.

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr
URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18



  De : Jobichen Chacko <jobich...@gmail.com>
 À : CCP4BB@JISCMAIL.AC.UK 
 Envoyé le : Mardi 6 mars 2018 5h11
 Objet : [ccp4bb] Off-topic question
   
Dear All,
We are trying to identify/sequence a DNA/RNA fragment (around 100bp) which was 
co-purified along with our protein.
The expression was done in E.coli.
Any suggestions on how to do this.
Thank you.
Jobi


   

[ccp4bb] Off-topic question

[Jobichen Chacko]

Dear All,
We are trying to identify/sequence a DNA/RNA fragment (around 100bp) which
was co-purified along with our protein.
The expression was done in E.coli.
Any suggestions on how to do this.
Thank you.
Jobi

Re: [ccp4bb] Off-topic question about SEC

[Nicolas RICHET]

Dear Reza,
Regarding the molecular weights (60 to 360 kDa), the low salt
concentration seems to drive the assembly of an hexamer.
Salt concentration could have a drastic effect on protein oligomerization.
If your A280 profile of the 360 kDa peak looks good (symmetric and in the
resolution limits), it is very likely to be an "hexamerization"!
As Nicolas FOOS said, you could confirm it using DLS or SEC-MALS (even
better).
Best wishes,
Nicolas




*Nicolas RICHET, Ph. D.Post-Doctoral ResearcherUniversity College Cork*
*School of Microbiology*

*Food Science and Technology Building*

*College Road*

*Cork City*
*IRELAND*


*Mobile: +353 (0)838385151*

2017-01-12 9:26 GMT+00:00 Nicolas FOOS <nicolas.f...@esrf.fr>:

> Dear Reza,
>
>
> in the past I had work with protein able to oligomerize reversibly but
> when oligomerization happened, even if I was able to separate and obtain
> monomeric protein,
>
> protein was not in a good condition.
>
>
> Have you try to characterize the two different states by DLS ? To
> discriminate "interaction with resin" that you suspect from oligomerization.
>
> Nicolas
>
>
> Nicolas Foos
> PhD
> Structural Biology Group
> European Synchrotron Radiation Facility (E.S.R.F)
> 71, avenue des Martyrs
> CS 40220
> 38043 GRENOBLE Cedex 9+33 (0)6 76 88 14 87 <+33%206%2076%2088%2014%2087>+33 
> (0)4 76 88 45 19 <+33%204%2076%2088%2045%2019>
>
> On 12/01/2017 01:50, Christopher Colbert wrote:
>
> What's your monomeric molecular weight?  Increased salt concentration can
> easily drive oligomerization.
>
> What is your evidence that it interacts with the resin?
>
> Cheers,
>
> Chris
>
> --
> Christopher L. Colbert, Ph.D.
> Associate Professor
> Department of Chemistry and Biochemistry
> North Dakota State University
> P.O. Box 6050 Dept. 2710
> Fargo, ND 58108-6050
> PH: (701) 231-7946
> FAX: (701) 231-8324
>
> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Reza
> Khayat <rkha...@ccny.cuny.edu>
> Reply-To: Reza Khayat <rkha...@ccny.cuny.edu>
> Date: Wednesday, January 11, 2017 6:42 PM
> To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK>
> Subject: Re: [ccp4bb] Off-topic question about SEC
>
> ​All these make sense. Protein is very strange cause it goes from 60kDa
> (globular) to an apparent 360kDa. Process is reversible too.
>
>
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
> --
> *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Keller,
> Jacob <kell...@janelia.hhmi.org>
> *Sent:* Wednesday, January 11, 2017 7:39 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Off-topic question about SEC
>
>
> Yes if it either
>
>
>
> A) oligomerizes
>
> B) significantly changes shape
>
> C) aggregates reversibly
>
>
>
> On option B: Lower NaCl could make the protein “appear” bigger by
> unfolding it a bit; hydrophobic interactions should be weaker in lower NaCl.
>
>
>
> JPK
>
>
>
>
>
>
>
>
>
>
>
> Artem
>
> www.harkerbio.com
>
> "where wild SEC columns roam free"
>
>
>
> On Jan 11, 2017 7:22 PM, "Reza Khayat" <rkha...@ccny.cuny.edu> wrote:
>
> Hi,
>
>
>
> Sorry for the off-topic question. Can a protein in lower [NaC] run faster
> on a SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The
> protein elutes well within the resolution limits of the SEC
> with a symmetric gaussian A280 profile. I know that at lower [NaCl] the
> protein can elute later because it may interact with the matrix.  Thanks.
>
>
>
> Best wishes,
> Reza
>
>
>
> Reza Khayat, PhD
>
> Assistant Professor
>
> City College of New York
>
> Department of Chemistry
>
> New York, NY 10031
>
>
>
>
>

Re: [ccp4bb] Off-topic question about SEC

[Nicolas FOOS]

Dear Reza,


in the past I had work with protein able to oligomerize reversibly but 
when oligomerization happened, even if I was able to separate and obtain 
monomeric protein,


protein was not in a good condition.


Have you try to characterize the two different states by DLS ? To 
discriminate "interaction with resin" that you suspect from oligomerization.


Nicolas


Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 12/01/2017 01:50, Christopher Colbert wrote:
What's your monomeric molecular weight?  Increased salt concentration 
can easily drive oligomerization.


What is your evidence that it interacts with the resin?

Cheers,

Chris

--
Christopher L. Colbert, Ph.D.
Associate Professor
Department of Chemistry and Biochemistry
North Dakota State University
P.O. Box 6050 Dept. 2710
Fargo, ND 58108-6050
PH: (701) 231-7946
FAX: (701) 231-8324

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK 
<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Reza Khayat 
<rkha...@ccny.cuny.edu <mailto:rkha...@ccny.cuny.edu>>
Reply-To: Reza Khayat <rkha...@ccny.cuny.edu 
<mailto:rkha...@ccny.cuny.edu>>

Date: Wednesday, January 11, 2017 6:42 PM
To: "CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>" 
<CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>>

Subject: Re: [ccp4bb] Off-topic question about SEC

​All these make sense. Protein is very strange cause it goes from 
60kDa (globular) to an apparent 360kDa. Process is reversible too.



Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

*From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK 
<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org <mailto:kell...@janelia.hhmi.org>>

*Sent:* Wednesday, January 11, 2017 7:39 PM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
*Subject:* Re: [ccp4bb] Off-topic question about SEC

Yes if it either

A) oligomerizes

B) significantly changes shape

C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by 
unfolding it a bit; hydrophobic interactions should be weaker in lower 
NaCl.


JPK

Artem

www.harkerbio.com <http://www.harkerbio.com>

"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" <rkha...@ccny.cuny.edu 
<mailto:rkha...@ccny.cuny.edu>> wrote:


Hi,

Sorry for the off-topic question. Can a protein in lower [NaC] run
faster on a SEC than at higher [NaCl] (i.e. elute at an earlier
volume)? The protein elutes well within the resolution limits of
the SEC with a symmetric gaussian A280 profile. I know that at
lower [NaCl] the protein can elute later because it may
interact with the matrix.  Thanks.

Best wishes,
Reza

Reza Khayat, PhD

Assistant Professor

City College of New York

Department of Chemistry

New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

[Christopher Colbert]

What's your monomeric molecular weight?  Increased salt concentration can 
easily drive oligomerization.

What is your evidence that it interacts with the resin?

Cheers,

Chris

--
Christopher L. Colbert, Ph.D.
Associate Professor
Department of Chemistry and Biochemistry
North Dakota State University
P.O. Box 6050 Dept. 2710
Fargo, ND 58108-6050
PH: (701) 231-7946
FAX: (701) 231-8324

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>>
Reply-To: Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>>
Date: Wednesday, January 11, 2017 6:42 PM
To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" 
<CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Off-topic question about SEC


​All these make sense. Protein is very strange cause it goes from 60kDa 
(globular) to an apparent 360kDa. Process is reversible too.


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Sent: Wednesday, January 11, 2017 7:39 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Off-topic question about SEC

Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by unfolding it 
a bit; hydrophobic interactions should be weaker in lower NaCl.

JPK





Artem
www.harkerbio.com<http://www.harkerbio.com>
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" 
<rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote:

Hi,



Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

[Keller, Jacob]

Whoa! Big change! Any possible physiological relevance?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Reza 
Khayat
Sent: Wednesday, January 11, 2017 7:43 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off-topic question about SEC


​All these make sense. Protein is very strange cause it goes from 60kDa 
(globular) to an apparent 360kDa. Process is reversible too.


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Sent: Wednesday, January 11, 2017 7:39 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Off-topic question about SEC

Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by unfolding it 
a bit; hydrophobic interactions should be weaker in lower NaCl.

JPK





Artem
www.harkerbio.com<http://www.harkerbio.com>
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" 
<rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote:

Hi,



Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

[Keller, Jacob]

Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by unfolding it 
a bit; hydrophobic interactions should be weaker in lower NaCl.

JPK





Artem
www.harkerbio.com
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" 
> wrote:

Hi,



Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

[Artem Evdokimov]

Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

Artem
www.harkerbio.com
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat"  wrote:

> Hi,
>
>
> Sorry for the off-topic question. Can a protein in lower [NaC] run faster
> on a SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The
> protein elutes well within the resolution limits of the SEC
> with a symmetric gaussian A280 profile. I know that at lower [NaCl] the
> protein can elute later because it may interact with the matrix.  Thanks.
>
>
> Best wishes,
> Reza
>
>
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
>
>
>

[ccp4bb] Off-topic question about SEC

[Reza Khayat]

Hi,


Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

[ccp4bb] off topic question about amount measurements of Membrane protein

[Yu, Kun]

Dear all,

I am a new starter in the membrane protein field and working with the
cell free expression system. As the really small amount of protein I
could get in hand, I got somehow stuck in the quantification of the
protein yield. so Is there any suggestion of any methods or reagents
suitable for quantifying small amount of membrane protein with
detergents or lipids? Before, I used spectrum meter to see Abs280 while
the reaction was scaled up to 200ul, but mostly I was playing around
25-50ul reactions and could only detected my proteins from western blot.

Many thanks!

Kun Yu

PhD Student at CSSB-FZJ, DESY
Notkestr. 85,
22607 Hamburg, Germany

Tel.: +49 40 8998 6032

[ccp4bb] off topic question regarding SPR

[Faisal Tarique]

Dear all

i have a basic question regarding SPR experiment and that is: What is the
recommended glycerol concentration in the protein sample for doing SPR
experiment and getting the best possible result.? i have kept 10% Glycerol
in my protein preparation..is it O.K ?

Thanx in advance

-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] off topic question regarding SPR

[Bosch, Juergen]

Hi Faisal,

whatever makes your protein happy is good. What type of experiment were you 
thinking of ? Protein-protein interaction or protein-small molecule interaction 
? In the latter case add some Tween20 or Brij-35 into the mix. A decent 
overview can be found under this webpage for what to consider when running your 
experiment. http://www.sprpages.nl
Since you are on this board, my assumption is you have crystallography-grade 
protein, which is an excellent starting point for SPR experiments. One thing to 
consider though is to perhaps include a glycerol correction, similar like a 
DMSO calibration curve, but that very much depends on what type of experiment 
you want to pursue.

Feel free to send me details off-board,

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu


On May 5, 2013, at 5:07 PM, Faisal Tarique wrote:

Dear all

i have a basic question regarding SPR experiment and that is: What is the 
recommended glycerol concentration in the protein sample for doing SPR 
experiment and getting the best possible result.? i have kept 10% Glycerol in 
my protein preparation..is it O.K ?

Thanx in advance

--
Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] off topic question BIAcore problem

[xianchi dong]

Dear all,

Recently I have measure a set of kinetic data of a receptor ligand
interaction using BIAcore 3000. To my surprise, the dissociation rate is
very low (~ 10e-6). During the measurement, I use a long dissociation time
(2 hours) .I repeat several time which give very similar results. So I am
wondering if the BIAcore can measure such a low off-rate kinetic. What is
the limitation of BIAcore? Any review about that?

Thanks in advance.

Xianchi Dong
Research Fellow
Children's Hospital Boston
Harvard Medical School

Re: [ccp4bb] off topic question BIAcore problem

[Bosch, Juergen]

I assume you use CM5 chips ?
I further assume you run at pH 7.5 perhaps ? What's the pI of your analyte ?  
7.5 ? Do you get significant binding to your reference cell under the 
conditions you are running ?
You might get rebinding to your negatively charged surface and the dissociation 
you are measuring might not really be correct (masked through rebinding)
Check that first I would say.
You can measure low picomolar dissociations. I recently had one with ~80 pM.

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 20, 2013, at 12:03 PM, xianchi dong wrote:

Dear all,

Recently I have measure a set of kinetic data of a receptor ligand interaction 
using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 
10e-6). During the measurement, I use a long dissociation time (2 hours) .I 
repeat several time which give very similar results. So I am wondering if the 
BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?

Thanks in advance.

Xianchi Dong
Research Fellow
Children's Hospital Boston
Harvard Medical School

Re: [ccp4bb] off topic question BIAcore problem

[Tomas Malinauskas]

Dear Xianchi,
unfortunately, dissociation rate constant kd 10^-6 s^-1 was just
beyond the limit of Biacore in 1999 (e.g. see Fig. 1 in
http://www.ncbi.nlm.nih.gov/pubmed/10556876). I am not sure about
these days.
As Juergen noted, you may have a problem with rebinding (you could try
to reduce amount of the ligand on the chip, see other tricks here
http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf).
Regarding reviews, David Myszka writes enjoyable reviews on SPR.
Hope that helps,
Tomas

On Wed, Feb 20, 2013 at 5:03 PM, xianchi dong dongxian...@gmail.com wrote:
 Dear all,

 Recently I have measure a set of kinetic data of a receptor ligand
 interaction using BIAcore 3000. To my surprise, the dissociation rate is
 very low (~ 10e-6). During the measurement, I use a long dissociation time
 (2 hours) .I repeat several time which give very similar results. So I am
 wondering if the BIAcore can measure such a low off-rate kinetic. What is
 the limitation of BIAcore? Any review about that?

 Thanks in advance.

 Xianchi Dong
 Research Fellow
 Children's Hospital Boston
 Harvard Medical School

Dea

Re: [ccp4bb] off topic question BIAcore problem

[Zhijie Li]

Hi Xianchi,

First of all: a very slow dissociation rate can also be an artifact: the 
analyte can be simply precipitating on the surface. You do need to rule this 
out by proper controls. 

But there is no rule saying that 10e-6 s-1 off rate is not realistic. Even with 
protein-small molecule binding, one can get extremely slow dissociation if the 
interaction is very strong. For example, the dissociation of biotin from 
streptavidin has a rate constant of the 10e-6 s(-1) order. A very slow 
dissociation rate is often (if not always) correlated with very tight binding 
(for example KD in picomolar range or even smaller). What is your calculated 
KD? The binding phase of the BIAcore curves should also reflect the fact that 
the off rate is low (the Kon-obs has an off rate term).

We have measured some pico molar bindings with BIAcore in the past. It is 
doable, but difficult. You may also want to consider in-solution methods (so 
that you do not need to worry about artifacts caused by the surface), such as 
ITC (by competition method), or fluorescence-based methods. 

For BIAcore, when working with very strong bindings (KD 100pM), there are a 
few things to consider:
1) You may find great difficulties regenerating the chip - probably the biggest 
concern with BIAcore (considering the ridiculous price of chips).
2) How much RU should you conjugate to the chip? With strong interactions, as 
low as possible amount of your ligand should be labeled on the chip, for 4 
purposes: a) to make regeneration easier; b) to reduce rebinding effect; c) to 
reduce mass transfer effect; d) to make sure you do not take away significant 
amount of analyte from the solution (discussed in 3)).
3) If you plan to span the KD range with the analyte, then if the KD is in 
picomolar molar range, you are supplying the surface with extremely dilute 
analyte solutions. In such case, you need to calculate if your flow rate is 
high enough, to compensate the loss of solute due to binding to surface, 
otherwise the real concentration of the analyte in the mobile phase will be 
much lower than the assumed analyte concentration.
4) The association phase of the BIAcore experiment is also affected by the 
dissociation rate.  The observed binding rate Kon-obs contains a Koff term. 
When you are loading the analyte at near KD concentrations, the binding will 
take similar amount of time as the dissociation phase to reach plateau. The 
Kon-obs also contains a concentration terml, so the time required for reaching 
plateau will be shorter and shorter when you load with higher concentrations of 
the analytes.
5) The only proper way of labeling chip for slow dissociations is by covalent 
means.

HTH,

Zhijie




From: xianchi dong 
Sent: Wednesday, February 20, 2013 12:03 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off topic question BIAcore problem


Dear all, 


Recently I have measure a set of kinetic data of a receptor ligand interaction 
using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 
10e-6). During the measurement, I use a long dissociation time (2 hours) .I 
repeat several time which give very similar results. So I am wondering if the 
BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?


Thanks in advance.


Xianchi Dong
Research Fellow 
Children's Hospital Boston
Harvard Medical School

Re: [ccp4bb] off topic question BIAcore problem

[xianchi dong]

Thanks a lot for the kind reply.

I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein
but I have different truncations of my protein which behaved similarly.
I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon
is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I
used was from 100 nM to 5 nM. I  used a control buffer which can disrupt
the binding. In that buffer, no binding was observed.

I also used fluorescence anisotropy to measure the Ki of the interaction
which showed a 1 nM binding compared with the 0.2 nM binding in the SPR
assay.


On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.comwrote:

 Dear all,

 Recently I have measure a set of kinetic data of a receptor ligand
 interaction using BIAcore 3000. To my surprise, the dissociation rate is
 very low (~ 10e-6). During the measurement, I use a long dissociation time
 (2 hours) .I repeat several time which give very similar results. So I am
 wondering if the BIAcore can measure such a low off-rate kinetic. What is
 the limitation of BIAcore? Any review about that?

 Thanks in advance.

 Xianchi Dong
 Research Fellow
 Children's Hospital Boston
 Harvard Medical School

Re: [ccp4bb] off topic question BIAcore problem

[Bosch, Juergen]

Well that looks pretty real then. You might have wrong concentrations in one or 
the other experiment perhaps hence the difference.

Jürgen

On Feb 20, 2013, at 5:45 PM, xianchi dong wrote:

Thanks a lot for the kind reply.

I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I 
have different truncations of my protein which behaved similarly.
I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is 
about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used 
was from 100 nM to 5 nM. I  used a control buffer which can disrupt the 
binding. In that buffer, no binding was observed.

I also used fluorescence anisotropy to measure the Ki of the interaction which 
showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay.


On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong 
dongxian...@gmail.commailto:dongxian...@gmail.com wrote:
Dear all,

Recently I have measure a set of kinetic data of a receptor ligand interaction 
using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 
10e-6). During the measurement, I use a long dissociation time (2 hours) .I 
repeat several time which give very similar results. So I am wondering if the 
BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?

Thanks in advance.

Xianchi Dong
Research Fellow
Children's Hospital Boston
Harvard Medical School


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] off topic question BIAcore problem

[Zhijie Li]

Hi Xianchi,

How did you treat the control flow cell? And what is the composition of the 
control buffer that you use to disrupt binding? Is it denaturing or a mild 
condition? 

If the Rmax=150 and Koff=1e-6, then at 7200 s (2 hr), the signal will only drop 
1RU from 150RU (assuming you can reach the Rmax), is that what you see on the 
dissociation curve? In reality, when loading analyte at 100nM, assuming you 
have Kon=1e4, Koff=1e-6, binding for 30min would only let you reach ~125 RU, 
and at the lower concentrations will be much worse. Consequently the total 
dissociation after 7200s would be less than 1 RU on most curves - I am not sure 
if the Biacore 3000 will have a stable enough baseline for you to confidently 
measure that. If you try to fit the dissociation phase alone to get the Koff, 
the reported Koff won't be too accurate due to the low S/N ratio. On the other 
hand the 5-100nM spanning would generate significant changes on the binding 
phase, thus the global fitting should be able to get a relatively accurate KD. 
0.2nM from SPR and 1nM from in-solution experiment also sounds reasonable.

Zhijie



From: xianchi dong 
Sent: Wednesday, February 20, 2013 5:45 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] off topic question BIAcore problem


Thanks a lot for the kind reply. 


I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I 
have different truncations of my protein which behaved similarly. 
I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is 
about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used 
was from 100 nM to 5 nM. I  used a control buffer which can disrupt the 
binding. In that buffer, no binding was observed. 


I also used fluorescence anisotropy to measure the Ki of the interaction which 
showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. 



On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.com wrote:

  Dear all, 


  Recently I have measure a set of kinetic data of a receptor ligand 
interaction using BIAcore 3000. To my surprise, the dissociation rate is very 
low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) 
.I repeat several time which give very similar results. So I am wondering if 
the BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?


  Thanks in advance.


  Xianchi Dong
  Research Fellow 
  Children's Hospital Boston
  Harvard Medical School

Re: [ccp4bb] Off topic question. NACCESS

[VAN RAAIJ , MARK JOHAN]

Dear Armando, 
don't know about NACCESS, but I guess it is superseded by AREAIMOL in CCP4 
(also in CCP4i); it outputs the accessible volume per atom in the pdb file and 
per residue and per chain and some other statistics in the log-file. 
Mark 

Quoting Armando Albert:

 Does anyone has got some information about how to get a mac version 
 (intel), of the old unix program naccess?. It was meant  to calculate 
 the solvent accessibility per residue from a pdb file.
 Armando

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es 

Re: [ccp4bb] Off topic question. NACCESS

[Iain Kerr]

Hi Armando,

The information can be found here:

http://www.bioinf.manchester.ac.uk/naccess/

It looks like you'll need to compile it from source code. I can help you 
with this, if you're unsure.


It also plugs in nicely to the excellent LIGPLOT, which I have found 
incredibly useful (and, by extension, HBPLUS).


best,
Iain

Iain Kerr
Senior Scientist
Dept. of Cellular  Molecular Pharmacology and
The Sandler Center for Drug Discovery
Phone: 415-502-8219 Fax: 415-502-8193
E-mail: ik...@cmp.ucsf.edu
QB3/Byers Hall Rm. 509
1700 4th Street
University of California
San Francisco, CA 94158
http://sandler.cgl.ucsf.edu/



On 6/19/2011 2:09 PM, VAN RAAIJ , MARK JOHAN wrote:

Dear Armando,
don't know about NACCESS, but I guess it is superseded by AREAIMOL in 
CCP4 (also in CCP4i); it outputs the accessible volume per atom in the 
pdb file and per residue and per chain and some other statistics in 
the log-file.

Mark

Quoting Armando Albert:

 Does anyone has got some information about how to get a mac version
 (intel), of the old unix program naccess?. It was meant  to calculate
 the solvent accessibility per residue from a pdb file.
 Armando

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es

Re: [ccp4bb] Off topic question. NACCESS

[Jacqueline Vitali]

I think one of the options of PISA is to calculate accessibilities -- I
recall when I was learning how to use pisa that I saw this option.

Also DSSP outputs accessibilities per residue

Areaimol in ccp4 does too.

I have seen many programs in the internet, google accessibilities,
accessible surface area.  what_if does something like this too.

On Sun, Jun 19, 2011 at 5:09 PM, VAN RAAIJ , MARK JOHAN 
mjvanra...@cnb.csic.es wrote:

 Dear Armando,
 don't know about NACCESS, but I guess it is superseded by AREAIMOL in CCP4
 (also in CCP4i); it outputs the accessible volume per atom in the pdb file
 and per residue and per chain and some other statistics in the log-file.
 Mark

 Quoting Armando Albert:

  Does anyone has got some information about how to get a mac version
  (intel), of the old unix program naccess?. It was meant  to calculate
  the solvent accessibility per residue from a pdb file.
  Armando

 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoléculas
 Centro Nacional de Biotecnología - CSIC
 c/Darwin 3, Campus Cantoblanco
 28049 Madrid
 tel. 91 585 4616
 email: mjvanra...@cnb.csic.es

Re: [ccp4bb] Off topic question. NACCESS

[Francois Berenger]

On 06/17/2011 05:25 PM, Armando Albert wrote:

Does anyone has got some information about how to get a mac version (intel), of 
the old unix program naccess?. It was meant  to calculate the solvent 
accessibility per residue from a pdb file.
Armando


Maybe this server can do the job:
http://cgal.inria.fr/abs/Vorlume/

And many other servers will be proposed maybe.

Regards,
F.

[ccp4bb] off topic question

[gauri misra]

Hi,
A nuclear receptor is purified only in the presence of strong affinity bound
ligand.
Is there some method to study and quantitate binding affinities of this
protein with other ligands (it is bound to the high affinity ligand after
purification)?
Attempts to purify in presence of low affinity ligands and subsequent
substitution trials were not successful.
I therefore seek suggestions from the expert community.


Gauri

[ccp4bb] Off topic: Question about CD spectrum

[P Hubbard]

Hi all,

I've limited experience interpreting CD spectra, so I might be missing 
something, but I've collected CD data for a hydrophobic 15-mer peptide that 
forms a putative alpha-helix. I get a region of strong negative ellipticity, 
bottoming out at ~222nm; however, I also get a strong positive peak (of equal 
magnitude to the 222nm minimum) at ~208nm. This 208 peak is opposite to a 
textbook alpha-helical spectrum (like it's been inverted).

From what I've read, short alpha-heical peptides often lack 208nm minima, and 
protonated carboxyl groups producce positive ellipticity at this wavelength; 
however, apart from the C-terminus, I don't have any carboxyl groups, and the 
peptide is in standard PBS buffer to boot. Any suggestions on what might be 
causing this odd 208 peak? NOTE: current data isn't below 200nm.

Thanks!

_
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[ccp4bb] Off topic question

[Gina Clayton]

Hi CCP4ers

 

Apologies for the off topic...

 

I have been trying to get hold of L-ribulose-5-phosphate (D- is readily
available). Does anyone know where I can get such a compound?

 

Thanks in advance for any suggestions and Merry Xmas!

 

 

Gina