Hi, Does high salt concentration(around 0.7M Nacl) in NiNTA elution do better. And what about the purification of high positively charged protein (like PI 10) in NiNTA.
Thanks Debajyoti Dutta On Sat, 28 Jun 2008 Artem Evdokimov wrote : >Ni salt of dodecyl sulphate is not soluble. Therefore (at least in theory) >SDS may leach the Ni out of the chelate and deposit it throughout the column >in baby-blue soapy flecks. Having said that, I must add that if you have to >use more than 0.1% SDS then you're dealing with a truly extreme case! > >Arginine at high concentrations can interfere with the chelaiton - pretty >much any amine and amino acid would do that in high enough amounts. I've >used arginine with Ni-NTA and His-Select resins and it wasn't an issue but >my concentrations were moderate (under 50 mM). For the same reason, high >concentrations of TRIS are not recommended either. > >Artem > >-----Original Message----- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jose >Antonio Cuesta-Seijo >Sent: Friday, June 27, 2008 5:37 PM >To: CCP4BB@JISCMAIL.AC.UK >Subject: [ccp4bb] Binding to Nickel in the presence of SDS or arginine > >Dear CCP4BBers, > >One of those questions regarding purification rather than >crystallography: >Reading the Qiagen manual for the Ni-NTA matrices, in the table of >compatibility of reagents with Ni-NTA matrices, SDS is mentioned >(only together with sarkosyl) as "Not recommended, but up to 0.3% has >been used successfully in some cases". Google is failing me to find >those cases, since pretty much every paper mentioning Ni binding also >mentions SDS-PAGE. Also, arginine is mentioned just as "not >recommended". >Why are SDS and arginine "not recommended", what are the physical and >chemical underlying problems? >Can they be used at low concentration without damaging the matrix or >abolishing binding? >Which are the maximal concentrations people had success with? > >Thanks a lot, > >Jose Antonio Cuesta Seijo > > > >************************************** >Jose Antonio Cuesta-Seijo >Cancer Genomics and Proteomics >Ontario Cancer Institute, UHN >MaRS TMDT Room 4-902M >101 College Street >M5G 1L7 Toronto, ON, Canada >Phone: (416)581-7544 >Fax: (416)581-7562 >email: [EMAIL PROTECTED] >**************************************
