Re: [ccp4bb] offtopic: related to protein purification
Dear Sonia, Have you passed the protein through Size exclusion column and confirmed that it contains a single oligomer? Do you use fresh protein preparation for ITC? If you have tried the above two, then increase the protein concentration. Checking at diff. pH buffer or temperature can also refine your data to a great extent. good luck On Mon, Aug 4, 2014 at 12:14 PM, Sonia Majumdar sonia@gmail.com wrote: Dear all, I am working on a GTPase with two tandem G-domains. While doing ITC experiments the heat change obtained is very poor 0.1ucal/sec, however the ITC profile is showing gradual saturation. The n value is very poor 0.33. I presumed the protein has bound GDP and tried to separate the nucleotide bound and unbound forms using Mono-Q. 3 peaks were obtained containing the same protein. However, ITC done with the proteins corresponding to the 3 peaks gave the same results. Please suggest what could the three peaks possibly mean and how to modify the ITC experiments. Thanks in advance. Regards. -- rashmi
Re: [ccp4bb] Thermofluor assay
Hi Theresa, Check if this works in your case http://www.sciencedirect.com/science/article/pii/S0969212608000609 good luck !! On Sat, Apr 12, 2014 at 4:38 PM, Theresa Hsu theresah...@live.com wrote: Dear all Does anyone has experience with Thermofluor assay to find the substrate transported/binding by a membrane protein? My protein does not have any similar structures and the substrate suggested by sequence analysis is not being transported in proteoliposome. I know ITC is good but I am looking for a more high-throughput way. Thank you. -- rashmi
Re: [ccp4bb] Very sad news: Guy Dodson
Prof. Guy Dodson He is one of the most wonderful person, I have met in my life. He had this very charming and vibrant personality, with whom one can speak about a very broad range of topics starting from Crystallography ... General Science ... Politics. His memory was just wonderful, I discussed some of my experimental issues with him during his visit to Bangalore, and we met again after 2 yrs in Perth and he still remembered me. That was such a special moment for me. It was my privilege to have known him. Guy and Eleanor are one of the sweetest couple to be seen. May his soul rest in peace. Rashmi On Wed, Jan 2, 2013 at 5:34 AM, Heather M. Baker h.ba...@auckland.ac.nzwrote: Guy Dodson 13-1-1937 to 24-12-2013 One of the most beloved and influential figures in the development of protein crystallography has died. Guy Dodson passed away peacefully on Christmas Eve, with Eleanor and other family members at his side. Together, Guy and Eleanor have enriched many lives. Many more tributes will follow, and the purpose of this short notice is just to make sure that this sad news reaches as many of their wide network of friends as possible. Born in Palmerston North, New Zealand (“the centre of the universe” as Guy put it), Guy went to Oxford in 1963 for postdoctoral studies with Dorothy Hodgkin and quickly became her right-hand person in the successful solution of the structure of insulin. These were exciting days, and set the stage for Guy’s subsequent establishment of wonderfully vibrant structural biology labs, first at the University of York and then at NIMR, Mill Hill. Many scientific successes followed, but for many of us our abiding memories are of Guy’s passion for life, and for science, his instincts for what was important, his great sense of fun, and his ability to make all of those who worked with him feel special. We all consider it a great privilege to have known him. We also wish to express publicly our support and affection for Eleanor and their wider family, who became part of many of our families, too. Ted and Heather Baker - on behalf of the New Zealand structural biology community. and all members of the York Structural Biology Laboratory.
[ccp4bb] off topic: ITC program error
Hi All Sorry for this off-topic, but I need an urgent help. We have an Origin 7 version of VP-ITC Microcal. I had shut it down 2 days back and switched it on today. When I am trying to open the VPviewer2000 where I can start my run it says that error as missing last inj. I had tried to run as administrator and it gives me the same message. Tried shutdown and restart, still the same error. Any expert advice about this is awaited. kind regards -- rashmi
[ccp4bb] sarkosyl
Hi All, I was wondering if anyone has used N-lauryl sarcosine in the lysis buffer for purifying protein and checked if the protein was functional or structurally okay Please let me know kind regards -- rashmi
Re: [ccp4bb] sarkosyl
Thanks Zhijie, So do you mean to say that your lab regularly uses it in Lysis and refolding buffers, and the protein/s purified are structurally and functionally fine and that there is no aggregation caused due to sarkosyl?? kind regards Rashmi On Thu, Sep 20, 2012 at 6:23 PM, Zhijie Li zhijie...@utoronto.ca wrote: ** Hi, We used it in some lysis/refolding buffers. Sigma marked this biodegradable detergent as Highly toxic by inhalation - when it's used in toothpastes and shampoos if I am not mistaken. when I called, Sigma said that since someone published a paper saying that when they sprayed it into a rat's nose the rat died, they had no choice but to put a skull sign on the MSDS. Zhijie *From:* Rashmi Panigrahi rashmi.panigrah...@gmail.com *Sent:* Thursday, September 20, 2012 5:47 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] sarkosyl Hi All, I was wondering if anyone has used N-lauryl sarcosine in the lysis buffer for purifying protein and checked if the protein was functional or structurally okay Please let me know kind regards -- rashmi -- rashmi
Re: [ccp4bb] off topic: ITC or Biacore
Thank you all for your suggestions. In the same way can Biacore work be done at lower temperature?? I want to do both ITC and Biacore for my protein and peptide regards Rashmi On Thu, Aug 9, 2012 at 9:19 PM, Edwin Pozharski epozh...@umaryland.eduwrote: On 08/09/2012 03:55 AM, rashmi panigrahi wrote: Does any one have the experience of doing ITC or Biacore(SPR) at 10 or 15 degrees? You can do ITC at any temperature your instrument allows, certainly no problems at 4oC. To share a trick, at least on a MIcrocal instrument cooling down is very slow, and I found it useful to wash the cell with ice cold water to quickly get down to 4 degrees. -- rashmi
[ccp4bb] off topic: protein peptide interaction using DSF
Hi all, Has anyone performed protein peptide interaction experiments using flurophore on a real time PCR machine? Please advice me on how the experiment is performed. Can I use ANS? with regards -- rashmi
[ccp4bb] protease inhibitor
Hi All, Does any one add protease inhibitor to purified protein just before setting trays? If yes what is the typical % that should be added ? regards rashmi
[ccp4bb] precipitation.
Hi all, 1) I have a protein which gives two peaks on the 1ml Histrap column, Has anyone seen this kind of behaviour and does this mean that there are two populations of protein. They are partially seperated. 2) I tried to load the two peaks seperately on the superdex-75pg column. They came out as roughly dimer but the difference in the peaks is 6mls According to calculation with gel filtration standards one was 1.8mer and the other was 2.3 mer Will there be problem if I mix the two peaks and load on the S-75 column?? 3) The protein is in 50mM HepespH7.3, 500mMKCl and 10% glycerol and imidazole when it comes from the NiNTA column, It is loaded on the superdex with same buffer but no imidazole. I get the dimer peak. If I concentrate and leave it, it start precipitating the next day even at 2mg/ml. I added bME (2mM) after the protein came out of superdex, and there was no precipitation. Hence for the next prep, I did the superdex run with bME in the buffer, there was a dimer peak and a small peak coming at the 125mls which is more than 1CV (S-75 is a 120 ml column). Loaded this small peak on the gel and it gave the same size band my protein. It could be my protein ... Hence I took the dimer from the above run concentrated and reloaded back on the same column and there was a very tiny peak for dimer which was 10 mAU for 0.5mls which is very less comapred to what I loaded. Does it mean that my protein is unfolded because of bME??? Any idea to stop precipitation would be helpful. with regards Rashmi
[ccp4bb] protein lost on membrane of centricon!!
Hi all, I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. I lost about 90% of my protein on the membrane of the centricon. Please suggest some way of concentrating this protein. Will concentrating using peg 20K be a good alternative?? regards rashmi
[ccp4bb] dissolving peptides !
Hello everybody, I have a 16 mer peptide which is predicted to be positively charged alpha helix and has 50% of its sequence hydrophobic. Could any one recommend the best way to dissolve it, and then it can be used for crystallization trials. thanks -- rashmi
[ccp4bb] Setting Microbatch trays !!
Hi, While setting up microbatch trays (under oil crystallization), 1. Has anybody used mineral oil (sigma M8410) and obtained some crystal hits? 2. Has anybody used anything other than Al's oil from Hampton, as Parafin oil from sigma or silicone oil from sigma and obtained some crystal hits? Thanks -- rashmi
