Re: [ccp4bb] ECM-29 - Abstract Deadline is Fast Approaching

And don't forget there are other MS to consider. And that Rovinj and that area are MOST agreeable holiday destinations! Exploited since Roman times.. Chairs: Navraj Pannu, Eleanor Dodson Recent devts in Phasing, Refinement and Autobuilding To cover: recent developments in phasing (including S-SA

Re: [ccp4bb] Protein expression

Hi Reza, In addition to the many useful suggestions already made, I would suggest lowering the final concentrations of IPTG. In many cases, 1mM IPTG interferes with expression levels and/or solubility. This suggestion does not address your concern for why things become ugly in going from 3mL to 50

[ccp4bb] Trouble with blot.

Dear all, sorry for off topic discussion. I am having trouble transferring protein onto PVDF membrane for N-terminal sequencing. When I used Tris-Glycine-Methanol in transfer buffer I got good blot, but when I used CAPS buffer (pH 11 set with 2N NaOH and 10% methanol) I did not get any blot. I tri

[ccp4bb] Two postdoctoral positions available in the Department of Medical Biochemistry and Biophysics (Karolinska Institute)

Dear CCP4ers, Sorry for this non-structural biology related jobs announcements, but this will be very helpful if you could spread this message to who might be interested. Two postdoctoral researcher positions are available in the Department of Medical Biochemistry and Biophysics at Karolinska I

[ccp4bb] Third CCP4-OIST workshop in Okinawa

Dear Colleagues, we are pleased to announce the third CCP4 structure solution school at the Okinawa Institute of Science and Technology (OIST), Okinawa. All details can be found at http://www.ccp4.ac.uk/schools/OIST-2015 Title: "CCP4 school: From data processing to structure refinement and bey

[ccp4bb] ECM-29 - Abstract Deadline is Fast Approaching

Dear all, at the ECM-29 in Rovinj, Croatia, there will be a microsymposium with the title "Structure solution on the fly - parallel data collection and structure analysis" The description of the MS reads like this: "Recent developments at synchrotron beamlines are enabling users to collect ve

Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

Thank you for your comments everyone. The CCP4BB is a wonderful resource and it has answered several questions that have been bothering me for years! Tristran has given us the correct conclusion as well as the important facts: the capacity of oil for holding O2 is high, but the diffusion rate is

Re: [ccp4bb] Protein expression

If you try to lyse a cell pellet from 500 ml via sonication compared to the 3 ml that is a huge difference. In particular if you don't scale up the total volume. Say you lyse your 3 ml culture in 10 ml and your 500 ml in 20 ml. Your local protein concentration is perhaps 50x higher then, which m

Re: [ccp4bb] Protein expression

Hello everybody, I’m posting this arguably off topic somewhat related to previous query question on behalf of a colleague: Does anyone have experience with Origami cells growing in fermenter? Many searches yield nothing as if these cells have never been used to grow in place other than flasks.

[ccp4bb] ECM29 microsymposium on Crystallization and Crystal Treatment

Dear All, just a reminder that the deadline to submit abstracts for ECM29 is Monday, bon weekend, Matthew. Dear All, The microsymposium on Crystallization and Crystal Treatment will take place at the the 29th European Crystallographic Meeting (ECM29), to be held August 23-28 in Rovinj, Croa

Re: [ccp4bb] Protein expression

Hi, Cultures are being properly cooled prior to induction (water bath supplemented with ice). Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070 From: lieh low [mailto:liehy...@gmail.com] Sent: Fr

Re: [ccp4bb] on Baton build

-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Smith, the starting position for the first baton is the centre of your screen, i.e. the pink box that usually resides there. I don't understand your second remark. If it is connected to the numbering, you can renumber the chain once it is conver

Re: [ccp4bb] Protein expression

Hi Reza, A few month ago I had a the exact same problem and we checked everything we could think of but without any improvement. Finally we were able to solve the problem only by subcloning the ORF into another plasmid (pET9a or pET29b). The big difference being the His tag position (C-ter or

Re: [ccp4bb] Protein expression

Reza, someone might have mentioned this, it takes longer time to cool down larger culture, we turn down the temp of the shaker when OD is about 0.4. For some protocol, we even use ice to cool the flask down before induction. You might also want to consider a lower induction temp, like 16degC. Maybe

Re: [ccp4bb] Protein expression

Hi, I’ve received a number of concurring suggestions. Some have requested more detail about the experiments. Here are the details. 1. Cells: BL21(DE3) 2. Plasmid: pET28a (T7 promoter) 3. Media: TB 4. Protein: cytoplasmic 5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with