Re: [ccp4bb] Off topic: His-tag purification

Hi Theresa: If you can make sure that your target protein is expressed. You can first use 6 M urea to denature the protein and then try to bind it to the column. If the denatured protein can bind to the column, it seems the histag is hided inside of the protein. It is not exposed enough to inte

Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

Hi Fred: For the mutated plasmids, it generates the nicked dna, so the transform efficiency will be lower compared to the parental plasmids. And that is the reason why people usually use super competent cell to transform these nicked plasmid. It seems ok to me to get 2 or 14 colonies from the

Re: [ccp4bb] On pKa of Aspartic acid

Hi Deepak: I think it is common for the residues which participate catalysis to have a Pka deviated from the reality pKa value especially for acid/base catalysis (acid base titration assay can help you to figure out the way of catalysis). Usually the pKa values of these kind of critical residu

Re: [ccp4bb] pH optimisation for crystallisation

Hello Sreetama: I think for crystallization, everything is hard to say. But if you find your crystal is sensitive to the pH, you certainly can optimize the pH value but it is better not to deviate a lot. For example you can make 0.2 unit interval (for example: pH value 4.5, 4.7, 4.9...etc which

Re: [ccp4bb] Dye for protein affinity measurement

Hello Jiahong: If I understand correctly that you want to test protein-protein interaction or inhibition study in solution, maybe you can try something like ELISA to test protein-protein interaction. Or if your B protein has 6 histag, you can use Ni-NTA agrose beads to test inhibition or bindi

Re: [ccp4bb] protein degradation

Hi Sivasankar: Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your prot

Re: [ccp4bb] about point mutation

Hello Arun: Actually, I am not sure about " i couldn't get my desire point mutation". You mean you didn't get the pcr product or you could get the pcr product but there is no mutation. If you didn't get the PCR,Just lower the annealing temperature to 55 degree. And try extension temperature 72

Re: [ccp4bb] Desalting columns

Hi Sangeetha: If you just want to check which buffer is good for your protein, maybe you can try to set up a crystallization screen, keeping your protein concentration just 3 mg/ml. You can observe (after several days) which conditions give you a clear drop, and maybe you can find a clue which

Re: [ccp4bb] no expression

Plus check the insoluble part or the whole cell. Date: Sat, 5 May 2012 16:30:06 -0400 From: jubo...@jhsph.edu Subject: Re: [ccp4bb] no expression To: CCP4BB@JISCMAIL.AC.UK Some very stupid questions from my side:- it is an expression vector and you are in frame with your gene of interest ?-

[ccp4bb] The binding between disordered and ordered proteins

Dear All: I have a general question about protein- protein interactions. I have two proteins, A and B. A is a disordered protein while B is a well folded protein. The binding between A and B has been approved by GST-pull down assay previously. The strange thing is I cannot get them bind if prot

Re: [ccp4bb] Biacore/SPR

Another option is BLItz which is cheaper and uses interferometry. Dee Date: Mon, 28 Oct 2013 11:05:33 -0400 From: jubo...@jhsph.edu Subject: Re: [ccp4bb] Biacore/SPR To: CCP4BB@JISCMAIL.AC.UK BiaCore 3000 or if you can afford the T200.Proteon XPR36 would be a cheaper Option and should work as w

Re: [ccp4bb] distinguish ligand binding sites within a protein

Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the p

Re: [ccp4bb] A question on protein microheterogenity for crystalization

Hi Acoot: Can your protein form some kind of dynamic self oligomers? When you test by using the gel-filtration column, if the peak is symmetry? Sometime if the target protein can have week self interaction, you can observe a tailed peak. If the protein can have a strong self interaction, maybe

Re: [ccp4bb] ITC with unfolded proteins

I have done once by using two proteins, one is disordered, the other is very well folded. The result I got is the baseline drift. The baseline goes up upon each injection. The reason I thought at that time is the heat capacity changed dramatically in the system. The disordered protein may form s

[ccp4bb] clashscore question

Hi All: I have a pdb file and the clashscores reported from both phenix and MolProbity were around 5, while the clashscore from the pdb validation server is 17. I wonder what may cause the difference? Any suggestion is appreciated. Thank you. Xiaodi ###

Re: [ccp4bb] clashscore question

ithout adding hydrogen to the structure. Regards Sharan On Tue, Jan 15, 2019, 11:23 PM Xiaodi Yu mailto:uppsala@hotmail.com> wrote: Hi All: I have a pdb file and the clashscores reported from both phenix and MolProbity were around 5, while the clashscore from the pdb validation

Re: [ccp4bb] model bias

Hi Aleks: Maybe you can try CNS ( Initial refinement by simulated annealing) also. It may help to get rid of the model bias and takes short time to run. Xiaodi > Date: Wed, 29 Apr 2015 13:52:53 +0200 > From: frederic.velli...@ibs.fr > Subject: Re: [ccp4bb] model bias > To: CCP4BB@JISCMAIL.AC.UK

[ccp4bb] Intra-molecular interactions

Dear All: I have a quick question: how common it is that electrostatic interactions are involved in intra-molecular interactions, particularly in intrinsically disordered proteins? Is this interaction specific and any example? Thanks, Dee Xiaodi Yu, Ph.D. Boston Children's Hospital &