Re: [ccp4bb] Off topic: room temp FPLC column cooling

Bear in mind that if you have cold buffers going into a room temperature (or warmer) pumps, you can get outgasing that will cause inaccurate flow or loss of priming for the pumps. You may be able to work around by using freshly degassed eluents, but the outgasing will still be a problem for long

Re: [ccp4bb] Off topic: room temp FPLC column cooling

Hi, a cheap solution would be to put buffers, fraction collector, and the column (if not too big) inside a cold cabinet, next to the FPLC. you probably need to use longer pipes but it works Best regards, GIA Le 07/06/2019 16:51, - - a écrit : Dear all, our cold cabinet FPLC is rotting

[ccp4bb] Off topic: room temp FPLC column cooling

Dear all, our cold cabinet FPLC is rotting away due to the high humidity in our lab. Anybody having a room temperature setup, where only the buffers, column(s) and fractions are being cooled? If so, by which means (cooling spiral, water pump, peltier element?). Thank you for your suggestions!

[ccp4bb] Off-topic - Alt-arrow shortcut keys on Formulatrix RockImager

Apologies for this off topic request but there may be someone on this list who can help. We have RockImager installed which uses Alt-arrow shortcut key to move between view of image settings (eg. UV and visible). This ability was lost a few months ago. I have asked Formulatrix who don’t know

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

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

[ccp4bb] AW: [ccp4bb] Off topic - French Press Accessories

ago, and everything's still working as perfectly as it did on the first day. best, j Von: CCP4 bulletin board Im Auftrag von Nazahiyah Rodzli Gesendet: Mittwoch, 24. April 2019 03:55 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Off topic - French Press Accessories Dear All, Our lab is using

[ccp4bb] AW: [ccp4bb] Off topic - French Press Accessories

. Best regards Miriam Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nazahiyah Rodzli Gesendet: Mittwoch, 24. April 2019 03:55 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Off topic - French Press Accessories Dear All, Our lab is using the good old Thermo French press (FA

[ccp4bb] Off topic - French Press Accessories

Dear All, Our lab is using the good old Thermo French press (FA-078A-picture attached) with a mini sample cell which has only 3.5 ml maximum sample capacity and planning to use for as long as it lasts. However, we are now in need of a bigger sample cell for our membrane protein work.

[ccp4bb] Off topic: Trouble Shooting on Expression of Membrane protein in Saccharomyces Cerevisiae

I was expressing Membrane protein (100 KD plant cell wall protein with N-terminal His tag) in Saccharomyces Cerevisiae. The vector i am using is PDDGFP modified vector which have URA selection and GAL1 as promoter. I used URA media and Galactose (2%) for induction. I have expressed this

Re: [ccp4bb] off topic: Membrane protein "into" soluble protein

[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew Lovering Sent: Wednesday, March 06, 2019 9:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic: Membrane protein "into" soluble protein Dear ccp4bb members, A quick off topic question. I have a protein whose domain structure

[ccp4bb] off topic: Membrane protein "into" soluble protein

Dear ccp4bb members, A quick off topic question. I have a protein whose domain structure runs N-TM-receptor-TM-enzyme-C, i.e. is a two transmembrane-helix containing signalling protein. One would suspect that the receptor domain has ends that cluster, and that the enzyme (via homology with

Re: [ccp4bb] Off topic question

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"

Re: [ccp4bb] Off topic question

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

Re: [ccp4bb] Off topic question

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"

Re: [ccp4bb] Off topic question

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

[ccp4bb] Off topic question

?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

[ccp4bb] off-topic: AUC facility

Hi everybody, We do not have access to a local analytical ultracentrifuge (Chicago area), and are in need of a reasonably priced facility for our urgent AUC needs. We are interested in KD's and thus sedimentation equilibrium. Recommendations are highly appreciated. Thank you, Engin --

[ccp4bb] off-topic: Laboratory RA job posting

Greetings Sorry for the off-topic post :) We are looking to fill an RA position in Enzymology/MolBio (junior or senior, depends on qualifications and experience). Full description follows. Interested candidates should forward their CV and letter of interest to: *i...@enkochem.com *

Re: [ccp4bb] OFF TOPIC

Hi Anamika, As far as I understood, the biotin in the elution buffer is helping your protein to get stripped off from the Avidin column. So, maybe you dialyze your purified protein (or run FPLC) and get rid of biotin completely, before you load the protein on to strptavidin

Re: [ccp4bb] OFF TOPIC

Hi Anamika, - Have you double-checked that the sequence of your cDNA is correct and includes the biotin acceptor peptide tag (BAP tag aka AviTag; GLNDIFEAQKIEWHE in single-letter amino acid code)? - Are you using a dedicated bacterial strain that over-expresses BirA enzyme? This may not be

Re: [ccp4bb] OFF TOPIC

otein Interactions From: CCP4 bulletin board on behalf of Anamika Singh Reply-To: Anamika Singh Date: Tuesday, 13 November 2018 at 10:15 To: "CCP4BB@JISCMAIL.AC.UK" Subject: [ccp4bb] OFF TOPIC Hi All, I am purifying the biotinylated protein (cloned into the pET28a vector) using

Re: [ccp4bb] OFF TOPIC

Hello, you probably purified a contaminant. Do a blot with an anti-biotin antibody or get electro-spray mass spectrometry done in order to confirm the identity of your protein. Wim On 13/11/2018 11:13, Anamika Singh wrote: Hi All,

[ccp4bb] OFF TOPIC

Hi All, I am purifying the biotinylated protein (cloned into the pET28a vector) using Avidin beads. Since I need the protein for SPR but when I used the purified protein to interact with Streptavidin coated onto the SPR chip. There was no signal. Can anybody tell me why is it so or how can I make

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

r 27, 2018 5:41 AM To: Whitley, Matthew J Cc: ccp4bb Subject: Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice Dear Matthew, I am also late in responding to this, but as part of a Nature Protocols paper on iMosflm (Supplementary Information for Na

Re: [ccp4bb] Off-topic, protein in dye-front (ion front?) on native-PAGE

Hi All, Sorry to bring this old topic up again. I planned to run tricine gels but I found a possible error in table 2 (4% stacking gel formula) in Hermann Schägger protocol (Nature Protocols volume 1, pages 16–22 (2006), the author wrote 3ml 3X gel buffer in a total of 12 ml solution, it should

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I am also a bit late in responding, I have a few incommensurately modulated protein crystal datasets that you would be welcome to use in your course. It would be neat for students to at least know that this type of diffraction exists. As far as I know, they can only be processed

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear Matthew, I am also late in responding to this, but as part of a Nature Protocols paper on iMosflm (Supplementary Information for Nature Protocols 12, 1310-1325, 2017) I provided a number of examples of “problem datasets”. Some of these are just two images, to show

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

For some reason, the September 19th ccp4bb digest got caught in my spam filter and didn't come through until a few minutes ago, so I didn't see several responses concerning interesting datasets for processing until just now. Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I'm a little late to the thread, but I thought I would still like to add DPF3b, kindly provided by Wolfram Tempel. This dataset is available on zenodo and forms the basis of a tutorial for using DIALS:

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear colleagues, I want to thank the following people for providing suggestions and comments about ‘difficult’ datasets suitable for teaching data processing: Tim Craig Jacob Keller Graeme Winter Aleksandar Bijelic Clemens Vonrhein Loes Kroon-Batenburg James Holton If anyone else has

[ccp4bb] Off Topic: tabletop ultracentrifuge

Hi, We are looking to get a tabletop ultracentrifuge. There are two companies to choose from, Beckman and Thermo Scientific (Hitachi). I have positive experience with the older Beckman model, TL-100, that I used long time ago. However, we don’t have any experience with the new Beckman models

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

It was brought to my attention that the link to the preprint I provided below doesn't work, but this one does: https://www.biorxiv.org/content/early/2018/08/18/394965 Thanks to Folmer Fredslund for pointing this out to me! -James Holton MAD Scientist On 9/21/2018 3:50 PM, James Holton wrote:

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi James, you’re probably aware of this but you can edit CBF headers in place with sed. That’s what I do when I make the detector on my diffractometer go closer than the hardware limit. All best - Andreas On Sat, 22 Sep 2018 at 00:51, James Holton wrote: > For teaching purposes I have found

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

For teaching purposes I have found that controlled pairs of data sets are most instructive.  You are right that an easy one-button-push processing run tells you nothing, but so does a bang-it-crashed-now-what data set.  Most useful are two data sets that are identical in every respect but one,

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear Matthew, In my search for the validity of meta data, I went through several data sets in SBGrid and proteindiffraction.org (IRRMC), especially those where automatic processing did not succeed are gave different results with different processing software. We reprocessed those data sets

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I have some notes which indicate that SBgrid data sets 5, 62, 78, 117, 218 posed problems for "automatic" processing using generate_XDS.INP / XDS when I saw them for the first time. Some of these problems (mainly the conversion of header values to ORGX ORGY) are taken care of in

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Matthew, One SBGrid example I used for a workshop was https://data.sbgrid.org/dataset/218/ This has the “wrong” beam centre as understood by (me/dials/xia2) which causes a certain amount of fun, nice example for use of the reciprocal lattice viewer and image viewer in DIALS to make sensible

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

istake, please reply to this message and follow with its deletion, so that we can ensure such a mistake does not occur in the future. -Original Message- From: CCP4 bulletin board On Behalf Of Whitley, Matthew J Sent: Wednesday, September 19, 2018 8:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject

[ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear colleagues, For teaching purposes, I am looking for a small number (< 5) of macromolecular diffraction datasets (raw images) that might be considered 'difficult' for a beginning crystallography student to process.  By 'difficult' I generally mean not able to be processed automatically by

[ccp4bb] [Off-topic] Job offer for a Postdoc position in Macromolecular Modelling and Design (MPI Dev. Biology)

Dear CCP4 community, In case any of you is interested in Computational Structural Biology, Protein Evolution and Protein Design, and is looking for a postdoc position, I share with you a job advertisement for a Postdoctoral Position in Macromolecular Modelling and Design at our department of

Re: [ccp4bb] Off-topic: protein 2d diagrams

Batch mode generation of residue-based diagrams of proteins Fabien CampagneEmmanuel BettlerGert VriendHarel Weinstein /Bioinformatics/, Volume 19, Issue 14, 22 September 2003, Pages 1854–1855,https://doi.org/10.1093/bioinformatics/btg236 Published: 22 September 2003 On 4-8-2018 10:38, Joana

Re: [ccp4bb] Off-topic: protein 2d diagrams

Try http://gpcrdb.org/ They have a tool for snake plots of GPCRs Best Gebhard Prof. Gebhard F.X. Schertler Structural Biology ETH Zürich D-BIOL Head of Biology and Chemistry Division Paul Scherrer Institut Laboratory of Biomolecular Research, LBR OFLC 109 CH-5232 Villigen PSI

[ccp4bb] Off-topic: protein 2d diagrams

Dear CCP4 community, I have seen many papers with detailed protein 2d diagrams like this one: However, I can never find a reference for any tool that can compute such diagrams from a 3D structure. Do you know of any? Many thanks and enjoy the warm weather! Joana — Dr. Joana Pereira

Re: [ccp4bb] Off-topic question

.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 D

Re: [ccp4bb] Off-topic question

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

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: FMLX RI1000 imager problems

Dear colleagues, slightly off topic question, but we have several issues with our Formulatrix imager and I would like to get some meanings of other users. If there are still some... ;-) Since some weeks we are loosing the interior LED storage illumination on startup. They lamps start

[ccp4bb] off topic: Add ligands/substrate directly into expression medium?

Hello everyone! In which condition can we add ligand/substrate directly to the expression medium for overexpression protein? One protein of i crystallized is a enzyme that catalyze sterols substrate, as we know the solubility of sterols is very poor, and the Km of the enzyme towards sterols

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

Perhaps this can be automated: https://www.phenix-online.org/papers/wd5073_reprint.pdf Software doesn't get tired or bored, and thus potentially can try more and produce more plausible interretations. Then one can hire a number of people of various expertise to choose "best" result according to

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

It sounds a bit like this paper from a year ago: https://www.nature.com/articles/ncomms12549 apart from the conclusion: here the point is that amateurs build higher quality models than crystallographers. On 20 November 2017 at 20:25, Shintaro Aibara wrote: > Dear All,

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

al message > From: Shintaro Aibara <shintaro.aib...@gmail.com> > Date: 20/11/2017 21:25 (GMT+01:00) > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] [Off-topic] Comparison of the same structure built by > many people > > Dear All, > > Apologies for the slightly

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

This one?http://scripts.iucr.org/cgi-bin/paper?SE0260 Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij Original message From: Shintaro Aibara <shintaro.aib...@gmail.com> Date: 20/11/2017 21:25 (GMT+01:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [Off-topic] Comp

[ccp4bb] [Off-topic] Comparison of the same structure built by many people

Dear All, Apologies for the slightly off-topic, but I was wondering if anybody knew of a paper/textbook where a protein model was built by multiple people (ranging from novice to experienced builders) and compared. I believe the conclusion was that while the overall trace was broadly correct,

[ccp4bb] off-topic: cell lysis using Microfluidizer method

Dear all, in our institute we thought about buying the Microfluidizer LM-20 for cell disruption (https://www.microfluidicscorp.com/microfluidizers/lab-machines/lm20/). It would be nice if you shared your knowledge with us concerning the handling, robustness, the frequency of repairs or spare

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

Hi Anamika, As it was said before, a cocktail of protease inhibitors should be fine. But let`s say you can not overcome the problem. It is not specified if you are doing this dialysis to cleave or not your His-tagged protein but even this you could try getting samples in 1 hour

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

suppose protease is the issue, then avoid overnight dialysis 发自网易邮箱大师 在2017年10月26日 22:18，Anamika Singh 写道： Dear All, I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris, 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the precipitation problem during

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

Hello, PMSF inhibits only 1 class of proteinases You can try to add other inhibitors : e.g. for purification from yeast I used the following in addition to PMSF: 1000 x stock individual inhibitors pepstatin5 mg/ml methanol chymostatin1 mg/mlDMSO antipain3 mg/mlmethanol (sonicate to

[ccp4bb] OFF topic (Protein degrades during Dailysis)

Dear All, I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris, 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the precipitation problem during dialysis but with the addition of 50mM L-Arg somehow managed to overcome the precipitation issue. But this time I

[ccp4bb] Off topic: Anyone still using Oxford Cryosystems Cryostream 600 series?

Dear colleagues, Anyone still using Oxford Cryosystems Cryostream 600 series? We would like to know if there is interest in a computer program to control these units via a rs232 connection. We are implementing this in our home source. For data collection, the program allows automated shutdown

Re: [ccp4bb] Off topic: denaturing urea gels

; Molecular Biology > The University of Chicago > pr...@uchicago.edu > > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher > Gileadi [opher.gile...@sgc.ox.ac.uk] > Sent: Saturday, September 30, 2017 3:44 PM > To

Re: [ccp4bb] Off topic: denaturing urea gels

C.UK Subject: Re: [ccp4bb] Off topic: denaturing urea gels In addition to the previous suggestions: With very small gels, the sample composition and depth (in the well) have a strong effect on the resolution. Rinse the wells with TBE buffer just before loading, as urea from the gel diff

Re: [ccp4bb] Off topic: denaturing urea gels

In addition to the previous suggestions: With very small gels, the sample composition and depth (in the well) have a strong effect on the resolution. Rinse the wells with TBE buffer just before loading, as urea from the gel diffuses into the well and may prevent the sample from settling at the

Re: [ccp4bb] Off topic: denaturing urea gels

Smith: One should expect to see ladders each separated by 1 nt in such cases. Mohammed: My suggestions: 1) running at 70V seems low. I do not see a reason for not using higher voltages. 200Vx45min should be OK. IT is better to not let the BPB front run out of the gel - so that you know whether

Re: [ccp4bb] Off topic: denaturing urea gels

your enzyme cannot give definite fragment. thus smear 发自网易邮箱大师 在2017年09月30日 19:28，Mohammad Khan 写道： Dear all, I am working with an exonuclease and I run the digested DNA on a 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system and cast gels of about 8.6x6.5 cm

[ccp4bb] Off topic: denaturing urea gels

Dear all, I am working with an exonuclease and I run the digested DNA on a 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 15 well comb. I run my gels at 70 V for as long as 4 hours till my

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

t; Khan <mohdkhan0...@gmail.com> > *Reply-To: *Mohammad Khan <mohdkhan0...@gmail.com> > *Date: *Friday, 21 July 2017 at 14:32 > *To: *"CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> > *Subject: *[ccp4bb] Off topic: Flourescence anisotropy measurement >

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear Didier and Opher, The difference in polarities can reach upto 60 units. I have tried concentrations between 1-5 nM DNA. Maybe I will give higher concentrations a shot. Thanks! On Fri, Jul 21, 2017 at 4:02 PM, Didier Spittler wrote: > Hello, > > What is your

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear Julius, That is a good suggestion. I will definitely try this. Thanks! On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius wrote: > Dear Mohammad, > your buffer is key to success in biophysical measurements. While your > protein may be totally fine in standard buffer, the

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

, 2017 9:44 AM To:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement Completely agree, you need a higher DNA concentration. We have had luck with 10 nM DNA. Also, bubbles have a HUGE impact on how the fluorescent signal is measured. Make sure you spin yo

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

r...@uchicago.edu> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton [eilise.mil...@gmail.com] Sent: Friday, July 21, 2017 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement Comple

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

if one has multiple species , e.g. bound and free, with each a different relative brightness and anisotropy/polarisation, then the use ( and interpretation) of anisotropy is straight forward; just a sum weighted by molecular fraction and the relative brightness. For polarisation is far more

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

>Ideally you should convert polarization to anisotropy. Simple enough – but >some referees can get picky… What is the argument for anisotropy being better? JPK

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad Khan Sent: Friday, July 21, 2017 9:33 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement Dear all, I am trying to measure the difference in polarization upon the binding of the DNA to my protein

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Completely agree, you need a higher DNA concentration. We have had luck with 10 nM DNA. Also, bubbles have a HUGE impact on how the fluorescent signal is measured. Make sure you spin your plates down (assuming you are using them) to remove any bubbles. We just had an undergrad read his anisotropy

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

>> on behalf of Mohammad Khan <mohdkhan0...@gmail.com<mailto:mohdkhan0...@gmail.com>> Reply-To: Mohammad Khan <mohdkhan0...@gmail.com<mailto:mohdkhan0...@gmail.com>> Date: Friday, 21 July 2017 at 14:32 To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>"

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

gmail.com> Reply-To: Mohammad Khan <mohdkhan0...@gmail.com> Date: Friday, 21 July 2017 at 14:32 To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement Dear all, I am trying to measure the difference in polariz

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Hello, What is your difference between the maximum and minimum value ? Have you try to change the probe concentration? Best, Didier Le 21 juil. 2017 15:34, "Mohammad Khan" a écrit : Dear all, I am trying to measure the difference in polarization upon the binding of

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

FP is the ratio between two fluorescence measurements; if the fluorescence signal is too low, you will still get a ratio but it will be essentially noise. Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in the low nM range, you may have to use other methods to

[ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all, I am trying to measure the difference in polarization upon the binding of the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in difference of polarization with decrease in protein

Re: [ccp4bb] off-topic: negative thermal shift upon ligand binding

In theory, what you say is quite sensible. But there is one interesting counter example I am aware of.The fragment tool compound that eventually gave rise to the clinical compound indeglitazar ( http://www.pnas.org/content/106/1/262.full.pdf) gives a negative shift by DSF (in our hands):

Re: [ccp4bb] off-topic: negative thermal shift upon ligand binding

ubject: [ccp4bb] off-topic: negative thermal shift upon ligand binding To: ccp4bb@jiscmail.ac.uk Hello, I am working on DSF to verify if some compounds bind to my protein. I see a negative shift of about 3-4 degrees upon ligand addition (dose-response) in comparison to the protein alone. I

[ccp4bb] off-topic: negative thermal shift upon ligand binding

Hello, I am working on DSF to verify if some compounds bind to my protein. I see a negative shift of about 3-4 degrees upon ligand addition (dose-response) in comparison to the protein alone. I assume that this might be due to the binding of compound to the unfolded stated rather than folded

Re: [ccp4bb] off topic

2017 7:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic Hi, Is anyone has worked with STAT1 proteins? I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was no expression so far or rather say inconsistent expression. Sometimes the expression was in inclusion

Re: [ccp4bb] off topic

be happy to help you. Cheers, Mirella Get Outlook for Android<https://aka.ms/ghei36> From: David Blum Sent: Thursday, 6 July, 22:10 Subject: Re: [ccp4bb] off topic To: ccp4bb@jiscmail.ac.uk Hi Anamika, I did a search and it looks like researchers are using either mammalian

Re: [ccp4bb] off topic

Hi Anamika, I did a search and it looks like researchers are using either mammalian cells or baculovirus to express this protein. I don't have experience with this particular construct so could you tell me why you choose *E. coli*? I run a protein expression facility and we typically use HEK or

Re: [ccp4bb] off topic

Hey Anamika I know SH2 domain is very much well-studied domain, and i am not sure why are you facing troubles in the expression of this protein. Just go through the literature and read about different protocol of expression of SH2 domain from several different proteins.. Well, reading that you

[ccp4bb] off topic

Hi, Is anyone has worked with STAT1 proteins? I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was no expression so far or rather say inconsistent expression. Sometimes the expression was in inclusion bodies. I have tried different methods to pull out the protein from

[ccp4bb] [off-topic] PhD position available

Dear all, A BBSRC funded PhD position is available in my laboratory in Exeter, focusing on enzymes for synthetic biology. The work is most likely going to be more enzymology than structural work, but please pass this on to any candidates who might be interested. Full details are available

[ccp4bb] Off topic but of interest to SSRL users

Dear All, For those that have Facebook accounts the SSRL Users Organization has established a Facebook page at https://www.facebook.com/ssrluec/. This has details on the SSRL/LCLS users meeting and news stories coming from SSRL. Recently you may or may not have heard that the US Department of

Re: [ccp4bb] off topic: high resolution animation in chimera

Update: to get transparent background in GIF: Chimera command file (now saves PNG with transparent background by adding "set bgTransparency"): close all;open #0 ABCD.pdb; set bgTransparency; ~disp;ribbon :.a; movie record directory "D:\mov_temp\" format png pattern frame_*; turn y 1

Re: [ccp4bb] off topic: high resolution animation in chimera

Hi Shanti, To get more control, you can use Chimera to generate the frames, save each of them as PNG, then process and re-assemble the frames in photoshop to make GIFs. The command line toolbox Imagemagick is also a great way for making GIFs (shown below). Step1 generating the frames

[ccp4bb] off topic: high resolution animation in chimera

Dear all, I am trying to make an animation using chimera. But it looks like that the animation output is poor quality, seems less dpi. How to get a high resolution animation, say 300 dpi, using ucsf chimera. I am using movie record commands. Thank you very much, Shanti Pal

Re: [ccp4bb] Off-topic: 3D printing tools for molecular models

Thanks, Edward. Yes, doing more manipulations with space-filling/surfaces is something I've been thinking about adding. There is less that can automated when things become more irregular and the meshes become more complex, however. These tools can be used to do some mixing of surface and

Re: [ccp4bb] Off-topic: 3D printing tools for molecular models

That is beautiful! The pins-and-holes approach might be useful also for space-filling models of multi-subunit complexes- both for holding the subunits together, and in cases where one subunit partially encircles another, the subunit would be sliced in half and pins could hold the halves

[ccp4bb] Off-topic: 3D printing tools for molecular models

Sorry for the somewhat off-topic post. After getting interested in 3D printing I quickly found that making nice ball-and-stick objects was very difficult to do on the typical fused filament printers most people can afford. This is largely because of the amount of support structures needed for

Re: [ccp4bb] off topic: MacPymol and macOS Sierra

Hi, I had this problem with Yosemite. Clicking on the icon still doesn’t open the program, but I can start it using the sh command in XQuartz.. I’m not sure if there was something else I had to do in order for this to work (it’s been a while since I started using it like this), but perhaps just

[ccp4bb] off topic: Change of bottom filter

Dear All I am wondering if its possible to change the bottom filter of the sepharose 12 gel filteration column. best Adriana

[ccp4bb] off topic: MacPymol and macOS Sierra

Hi all, Sorry for the very off topic message, but I recently upgraded my OS to Sierra and all of a sudden MacPymol has stopped working for me. Clicking on the program just gives the appearance of it about to start by showing the icon on the dock, and then just blinks out, without ever

Re: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

One possibility: surface-immobilised forms of the peptide HGGHHG bind strongly to His tags (coordinating around zinc rather than nickel). See https://www.ncbi.nlm.nih.gov/m/pubmed/15050643/. If you want true permanence, you could apply the trick used on BiaCore chips. They use a NTA-functional

[ccp4bb] Spam : Re: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

/labpages/computational_functional_genomics.html - Original Message - From: Jacob Keller <kell...@janelia.hhmi.org> To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 05 Apr 2017 08:49:40 +0530 (IST) Subject: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip Does anyone have a simple way to

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