[ccp4bb] pMAL p2X

2014-12-04 Thread rana ibd
Dear AllThank you for your infornation regarding this vectorBest RegardsRana


[ccp4bb] pMAL -p2X vector

2014-12-01 Thread rana ibd
Dear CCP4Sorry for the question, does anyone work with the pMAL-p2X vector not 
c2X, because I am trying to find this vector for a long time now, and neither 
NEB of life technologies provide this vector. Does anyone know from where  I 
can get it or any company provides it, I would be grateful.
Best RegardsRana


[ccp4bb] cryo-EM

2014-08-31 Thread rana ibd
Dear All
Thank you for your suggestions and articles regarding the cryo-EM 

Best Regards
Rana

[ccp4bb] cryo-EM

2014-08-29 Thread rana ibd
Dear CCP4
I wanted to ask has anyone tested 3D protein structure by cryo-electron 
microscopy? what is the suitable concentration required for this procedure
Best Regards
Rana


[ccp4bb] maltose binding protein

2014-03-27 Thread rana ibd
Dear CCP4 
Does anyone know how to remove the maltose binding
protein after cleavage from the target protein; I have tried gel filtration and
ion exchange but with no luck, my protein is interacting with the MBP even
after complete cleavage. I would be grateful for any help or suggestions 
Best Regards
Rana

Re: [ccp4bb] maltose binding protein

2014-03-27 Thread rana ibd
Dear Mark
Thank you for yor reply, and yes I have tried adding it to the maltose resin 
after cleavage but the MBP runs through with my protein, I have also tried 1M 
NaCl but with no luck and I also apply detergent after cleavage to the dialysis 
buffer because I usually dialyze after cleavage , is there anything that could 
maybe precipitate the MBP 
Best Regards
Rana





 From: Mark J van Raaij mjvanra...@cnb.csic.es
To: rana ibd rna19792...@yahoo.com 
Sent: Thursday, March 27, 2014 11:35 AM
Subject: Re: [ccp4bb] maltose binding protein
 

did you try the maltose-resin?
in principle it should bind MPB but not your protein. You can try to add salt 
or detergents to disturb interaction between MBP and your protein (also in gel 
filtration).
No guarantee of success, unfortunately not all protein behave nicely.

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij






On 27 Mar 2014, at 11:26, rana ibd wrote:

 Dear CCP4
 Does anyone know how to remove the maltose binding protein after cleavage 
 from the target protein; I have tried gel filtration and ion exchange but 
 with no luck, my protein is interacting with the MBP even after complete 
 cleavage. I would be grateful for any help or suggestions
 Best Regards
 Rana

[ccp4bb] Thank you

2014-03-27 Thread rana ibd
Dear CCP4 

Thank you for all your suggestions
Best Regards
Rana


[ccp4bb] cryoprotection condition

2014-02-08 Thread rana ibd
Dear CCP4
Does anyone know a good cryoprotection condition for these two conditions I 
would be grateful
The first condition:
 10% PEG 2, 20%PEG MME 550. 0.03M of NPS ( NPS is sodium nitrate, disodium 
hydrogen phosphate, and ammonium sulfate), 0.1MMES/Imadizole pH 6.5
The second condition:
20% PEG 3350, 0.2M sodium iodide or 0.2M sodium acetate
Thank you
Rana 

[ccp4bb] help

2013-08-21 Thread rana ibd




Dear ccp4
   I
wanted to ask if anyone has expressed DDB1 (Damaged DNA Binding protein)  or 
any other interacting partner using
bacterial cells instead of insect or mammalian or yeast cells, I also wanted to 
ask if
anyone has any experience with this binding partner interacting with a fusion
protein (containing an MBP tag) , I would be grateful for any ideas suggested
Best Regards
Rana 

Re: [ccp4bb] Protein concentration for crystallization

2013-06-10 Thread rana ibd
Dear Debasish 
What do you mean by percentage? do you mean consentration? so if you mean cons. 
I think you should test you protein using a TCP kit to observe at what cons. 
would your protein  precipitate, this way you would verify the convinient cons. 
for your protein before crystallization
Best Regards
Rana

From: Debasish Chattopadhyay debas...@uab.edu
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 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

[ccp4bb] protein cleavage

2012-11-04 Thread rana ibd
Dear CCP4
 I am having a problem with cleaving my fusion protein and I would be 
grateful if you advice me regarding this situation,  I have an MBP-DHBx fusion 
protein and I am trying to cleave it using TEV protease, I have tried different 
ratios and different temperatures  with different incubation time but still it 
will not cleave, all I observe on the gel is the bands of the fusion protein 
which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa 
and no sign of the DHBx which is 17kDa,I have also checked the sequence if 
there was any problem but I could not find anything unusual the sequence was 
fine , so if you have any suggestions regarding this situation I will be 
thankful 
Best Regards
Rana 

[ccp4bb] protein cleavage

2012-11-04 Thread rana ibd
Dear All 
   Thank you for all your replies, the buffer for the TEV protease that I have 
used contains 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, and 1mM DTT at PH= 8.0. I 
have tried using this buffer without NaCl but the TEV protease precipitates 
when dialyzing over night, as for using glutathione (reduced alone, also with 
the oxidized) still had the same result
Rana

[ccp4bb] Dear ccp4

2012-08-27 Thread rana ibd
Dear CCp4
Thank you for all your and information suggestions regarding question
Best Regards
Rana


[ccp4bb] Dear ccp4

2012-08-26 Thread rana ibd
Dear ccp4 
   I am working on hbx (Hepatitis B x protein for both that duck and the human) 
I worked first on the dhbx which  contained the vector pRSET-A and I didn’t get 
any results, then I changed the vector ( thanks to many of you who provided me 
with information, papers and books) using p-MAL c2x which worked perfectly 
throughout the whole procedure, I even now have about 60% soluble protein, then 
I started working on the hhbx,also changing the vector from pRSET-A to p-MAL 
c2xat first the PCR was successful with an obvious band on the gel, so I 
proceeded with double digestion using both restriction enzymes, incubated 3h 
then applied the insert on the gel, surprisingly what I observed was a slight 
band on the gel, after purifying the insert I checked the concentration which 
decreased from 35ng/ul before purification to 5.2ng/ul after purification, I 
would be grateful if any one has any idea or suggestions regarding this 
situation
Best Regards
Rana    

[ccp4bb] dear ccp4

2012-08-26 Thread rana ibd
Dear ccp4 
   I am working on hbx (Hepatitis B x protein for both that duck and the human) 
I worked first on the dhbx which  contained the vector pRSET-A and I didn’t get 
any results, then I changed the vector ( thanks to many of you who provided me 
with information, papers and books) using p-MAL c2x which worked perfectly 
throughout the whole procedure, I even now have about 60% soluble protein, then 
I started working on the hhbx,also changing the vector from pRSET-A to p-MAL 
c2xat first the PCR was successful with an obvious band on the gel, so I 
proceeded with double digestion using both restriction enzymes which are BamH1 
and Hind111, incubated 3h then applied the insert on the gel, surprisingly what 
I observed was a slight band on the gel, after purifying the insert I checked 
the concentration which decreased from 35ng/ul before purification to 5.2ng/ul 
after purification, I would be grateful if any one has any idea or suggestions 
regarding this situation
Best Regards
Rana    

[ccp4bb] dear all

2012-03-28 Thread rana ibd
Dear all 
yes my paalet is large enough and no my protein is not soluble ,and  I am using 
37degrees before and after induction with IPTG,and the vector that I am using 
is pRSETA 3.2kd
Best Regards
Rana




 From: Kelly Daughtry kellydaugh...@gmail.com
To: rana ibd rna19792...@yahoo.com 
Cc: CCP4BB@jiscmail.ac.uk 
Sent: Tuesday, March 27, 2012 5:45 PM
Subject: Re: [ccp4bb] dea all
 

When you lyse the cells and spin down cellular debris, is the pellet large and 
white (indicating inclusion bodies)? Is your protein soluble or membrane? What 
temperature did you use for expression? What vector are you using? Providing 
more details allows us to better answer your questions.

Off the top of my head:
Altering expression can include lowered the temperature just prior to induction 
(25, 18, or lower) and letting the cells grow overnight. Induction at increased 
cell density (1.0 vs 0.6 O.D.).

Anther option to increase the expression of soluble protein is to use the 
auto-induction media: http://www.ncbi.nlm.nih.gov/pubmed/15915565 

Another option is to try other cells lines, and co-expression 
with chaperonins (the arctic express cell line is useful 
http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=ProductSubPageType=ProductDetailPageID=467 
 ).

Did you try other tags (GST, MBP, etc)?

Hope this helps,
Kelly Daughtry

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***



On Tue, Mar 27, 2012 at 10:35 AM, rana ibd rna19792...@yahoo.com wrote:

Dear all
I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB 
madia, I am having problems with the expression which shows small amount of 
the protein , I also have problems with 
purification using NI-NTA by also having small amount even after 
extensive buffer exchange , Is it likely due to the small amount of 
protein in the medium , should I use a different kind of media, any 
sugestions or any kind of details or a paper that might help I will be 
thankful


Best Regards 
Rana