Re: [ccp4bb] Protein Ligand interaction using SPR technique

Dear Praveen, As said, Biacore3000 is not the best for small molecules, but in some case you can do nice things with this system. I see 3 possibilities : - You can immobilize your molecules on the chip if you have an amine on it which is supposed to not participate to the interaction. - You can

Re: [ccp4bb] protein precipitation reg

Please have a look at the very elaborated/detailed discussion by Martin Chaplin on chaotropes and kosmotropes http://www1.lsbu.ac.uk/water/kosmotropes_chaotropes.html and on the Hofmeister series http://www1.lsbu.ac.uk/water/hofmeister_series.html and *note the diference* in the way the ions

Re: [ccp4bb] Protein Ligand interaction using SPR technique

Hi Praveen, The BiaCore 3000 is an older model, and is not optimised for protein/small molecule interactions. The BiaCore T-100 and T-200 have enhanced sensitivity and are better suited small molecules (assuming you have to use SPR). As for chip choice, it depends an awful lot on the behaviour

[ccp4bb] Protein Ligand interaction using SPR technique

Dear all, Sorry for off-topic question. I want to study protein interaction with few small molecules using SPR (Machine- *BIACORE 3000*). The recombinant protein expressed in bacterial expression system is of 92 kDa (with His tag), pI= 9. *Question-* 1. What should be the chip of choice- *NTA

Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card under Windows 10

Hi All, I summarized my Coot and Pymol 3D experience here. Monitor is from Asus 24 or 27 inch 3D monitor. Monitor works with Nvidia 3D kit. Refresh rate set 120 HZ. Please first download and install Nvidia 3D kit driver and Asus monitor driver and install first when working with Windows 7 or 10

Re: [ccp4bb] protein precipitation reg

On 03/30/2017 08:10 AM, mesters wrote: If the pI of the protein is below the pH of the buffer (net negatively charged protein), optimum stabilization (salting out; lower solubility) of the macromolecule is achieved by combining a kosmotropic anion with a chaotropic cation, e.g.

Re: [ccp4bb] binding protein to nickel column

Hi Shubhangi, As Edward suggested, you can try with N-terminal His tag. For this you can either clone in N-terminal His tagged based vectors or by site directed insertion of 18 nucleotides coding for 6 His at the N-terminus region. But, before that, I

Re: [ccp4bb] binding protein to nickel column

Hi, Shubhangi, One possibility is that the his-tag could be non-accessible. Have you tried moving to the tag to the other end of the protein (N vs C)? Another thing that worked for me for a past project is using a small amount (1-2M) of urea for the IMAC step and then dialyzing away the urea

[ccp4bb] binding protein to nickel column

Hello I have a 10kda histidine kinase domain protein with a pI of 9.5. It has a C-term his tag and despite using different buffers the protein doesnt bind to the nickel cloumn. it comes out in the flow trhough. Buffers used- 50mM tris Ph=8, 300mM NaCl 50mM tris Ph=7, 300mM

Re: [ccp4bb] ligand with out space group information

On 30/03/17 14:59, chemocev marker wrote: Hi Hi. I have model of ligand molecule and it does not open in coot. Its not a crystal structure. I can view it in the pymol or chimera but not in the coot. It gives error that it does not have any space group information. Is there is a way to open

Re: [ccp4bb] protein precipitation reg

Yes, I once spent quite a bit of time engineering mutations into my target to improve solubility to exclude detergent from the purification to improve crystal growth and diffraction: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374227/ If you think buffer conditions hold the key to your

[ccp4bb] ligand with out space group information

Hi I have model of ligand molecule and it does not open in coot. Its not a crystal structure. I can view it in the pymol or chimera but not in the coot. It gives error that it does not have any space group information. Is there is a way to open it in coot. best Jiri

Re: [ccp4bb] protein precipitation reg

Hi Akila, I'm curious about your choice of pET22b. If you cut out the signal peptide that comes with the vector and express your protein in the cytoplasm, then what I am about to say doesn't apply. However, if you are exporting the protein into the periplasm, have you considered doing osmotic

Re: [ccp4bb] protein precipitation reg

If the pI of the protein is below the pH of the buffer (net negatively charged protein), optimum stabilization (salting out; lower solubility) of the macromolecule is achieved by combining a kosmotropic anion with a chaotropic cation, e.g. Ammoniumsulfate (most successful salt)! /For your pI

Re: [ccp4bb] protein precipitation reg

If I remember correctly, Triton X-100 (or any other surfactant for that matter) is a bad idea for protein intended for crystallography. I can't remember the paper, but I'm sure I read that somebody showed it's basically impossible to remove all of the surfactant molecules from the protein no

Re: [ccp4bb] Large number of outliers in the dataset

This case is encouraging to me that a structure can be solved with such high mosaicity (in your report is 1.9). I wonder how the diffraction looks like (I imagine spots smearing or streak). With such high mosaicity, the unit cell dimension and space group determination is highly likely not

Re: [ccp4bb] protein precipitation reg

I personally like TRIS for the first few steps of purification and then change to something else during my last dialysis step. I mostly work with bacteria and they often produce lysates that have pH's that are too acidic for good nickel affinity chromatography, which is why I use 100 mM TRIS pH

[ccp4bb] PSI Career Return program

Dear All, I would like to draw your attention to the career reintegration program at the Paul Scherrer Institute, Switzerland. The goal of the program is to restart the academic careers of scientists (Masters or postdoc) interrupted due to the family reasons, for example by having children or

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

yes - really, tris should be the buffer of last resort rather than the standard. its only general advantages would seem to be that it's cheap and not very toxic. j -- Professor Jon Hughes, BSc, PhD Institute for Plant Physiology Justus Liebig University, Giessen Zeughaus, Rm. 341

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

yes, "oil of vitriol" is sulphuric acid, "blue vitriol" is copper II sulphate as i recall. j -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von CRAIG A BINGMAN Gesendet: Donnerstag, 30. März 2017 05:52 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re:

Re: [ccp4bb] protein precipitation reg

Only one of your dialysis buffers has a decent (100 mM) NaCl. Maybe you could try higher salt IN COMBINATION with glycerol. There's also NDSBs that stabilise proteins. Also, could the high immidazole be keeping the protein happy? You could test this by dialysing into the same buffer with a

Re: [ccp4bb] protein precipitation reg

Dear all, I have used the following buffers for purification and dialysis. this is fyi. *Lysis buffer*: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl (increase in salt concentration increased precipitation of the protein in the column itself) *5mM Beta mercaptoethanol * *0.5% Triton X 100 * I