Re: [ccp4bb] stoichiometry of complex and incubator
Hi , yes as *Konstantin* said if u have cysteine residue in ur any one of protein u can label that protein with Pyrene malemide or oregon malemide or other fluorescence dye then remove the dye by desalting column. titrate with second protein and do fluorescence anisotropy experiment to get the KD value (stochiometry). one more thing u can do. u can add kinase motif in one of ur protein by re-cloning and then label with labelled ATP and then do EMSA with other protein. regards On Thu, Aug 4, 2011 at 9:13 AM, Konstantin v. Korotkov k...@u.washington.edu wrote: Hi Min, If your proteins have cysteine residues, or you could introduce them into the sequence, then you could label the interacting proteins with a fluorescent label, run an SDS-PAGE and quantify the bands with a fluorescent scanner. See for example Supplementary Figure 1C in Fronzes et al. (2009) Science 323: 266. -Konstantin On Wed, 3 Aug 2011, m zhang wrote: Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min -- Konstantin Korotkov, Ph.D. Research Scientist University of Washington Department of Biochemistry Box 357742 Seattle, WA 98195-7742 (206)616-4512 k...@u.washington.edu -- -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy India
Re: [ccp4bb] stoichiometry of complex and incubator
hi Min, I think SPR(Surface plasmon resonance) should suit your situation. The Biacore instrument I worked on required 200 µL of 10-50 µG protein(ligate) for immobilisation and 150µL of various concentration of the other protein(ligand) in the range of 50-1000 µg/ml. (depending on your situation the concentrations may vary) I hope you can use this technique. Best wishes, Isha Structural Biology Lab, SLS, JNU New Delhi On Thu, Aug 4, 2011 at 8:02 AM, m zhang mzhang...@hotmail.com wrote: Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min
Re: [ccp4bb] stoichiometry of complex and incubator
Dear Min, You can use microscale thermophoresis for this kind of tricky cases when you have limited amount of protein and ligands. There is enough literature on this topic. have a look in the following reference: Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19678-82 Hope this helps. Regards, Krishna Krishna Chinthalapudi Graduate Student Hannover Medical School Germany On Thu, Aug 4, 2011 at 4:32 AM, m zhang mzhang...@hotmail.com wrote: Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min
Re: [ccp4bb] stoichiometry of complex and incubator
Hi Min, Given the limited supply of your protein you could try the EDC-NHS zero-length crosslinking either in one-step or two-step protocol (Anal. Biochem. 185,131,1990). If your protein complex is like most protein complexes chances are there will be some electrostatic contacts at the interface (Glu-Lys or Asp-Lys). These can be crosslinked and you need only micrograms of your protein to test it. The caveat here is that crosslinked proteins usually do not migrate on SDS-PAGE according to their Mr., but you can get a pretty good idea from the pattern of crosslinking at various protein ratios and by changing the extent of crosslinking. You can also label one of your proteins with a fluorescent probe (SH or NH2 specific) and determine stoichiometry form the change in fluorescence/Coomassie staining of the crosslinked products as compared to the labeled monomer. Good luck Zenon
Re: [ccp4bb] stoichiometry of complex and incubator
Dear all, Thank you all very much for your kind suggestions. Now we have some preliminary data of SPR. But we might try other new ideas suggested here. Best,Min Date: Thu, 4 Aug 2011 13:19:41 +0200 From: krishna@gmail.com Subject: Re: [ccp4bb] stoichiometry of complex and incubator To: CCP4BB@JISCMAIL.AC.UK Dear Min, You can use microscale thermophoresis for this kind of tricky cases when you have limited amount of protein and ligands. There is enough literature on this topic. have a look in the following reference: Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19678-82 Hope this helps. Regards, Krishna Krishna Chinthalapudi Graduate Student Hannover Medical School Germany On Thu, Aug 4, 2011 at 4:32 AM, m zhang mzhang...@hotmail.com wrote: Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min
[ccp4bb] stoichiometry of complex and incubator
Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min
Re: [ccp4bb] stoichiometry of complex and incubator
Hi Min, If your proteins have cysteine residues, or you could introduce them into the sequence, then you could label the interacting proteins with a fluorescent label, run an SDS-PAGE and quantify the bands with a fluorescent scanner. See for example Supplementary Figure 1C in Fronzes et al. (2009) Science 323: 266. -Konstantin On Wed, 3 Aug 2011, m zhang wrote: Dear all, I have two unrelated questions. Any suggestion on any of them will be greatly appreciated. First, we have two proteins that bind each other without a doubt. But since we have very limited amount of proteins and it takes a long time to reproduce them, we are very hesitating to try ITC and AUC to find out the stoichiometry of these complex. So I am wondering if there is any other way we can try to find the ratio without consuming much proteins? Second, we are thinking about getting a new incubator for crystallization. Could anyone here recommend a model or a brand? Thank you, Min -- Konstantin Korotkov, Ph.D. Research Scientist University of Washington Department of Biochemistry Box 357742 Seattle, WA 98195-7742 (206)616-4512 k...@u.washington.edu --
