[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

[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 r

[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] stock solution of glutathione

Hi, I wanted to use glutathione for cocrystallizing with my protein at about 5mM concentration. What is the concentration of the stock solution to be prepared for crystallization studies. Do I have to make the stock solution of glutathione fresh each time or can I make the stock and store at -20de

[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? Tha

Re: [ccp4bb] Fwd: regarding purification of DNA binding protein

Dear Arpita, For Getting rid of DNA you can incubate the lysate with DNase and incubate it before lysing the cells with sonication or french press (or anyother method of your choice) cheers Rashmi On Wed, May 12, 2010 at 8:53 PM, Arpit Mishra wrote: > > > -- Forwarded message

[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

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 wrote: > Dear all > > Does anyone has experience with Thermofluor assay to find the substrate > transported/binding by a mem

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

[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

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 wrote: > > On 08/09/2012 03:55 AM, rashmi panigrahi wrote: >

[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

blished 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 > *Sent:* Thursday, September 20, 2012 5:47 AM > *To:* CCP4BB@JISCMAIL.AC.UK >

[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 adm

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 so