Thanks for the all the reply my protein is filgrastim and is biosimilar to Neupogen. it is stable at acidic pH of 4.0. i want to get rid of dimer before i formulate it as we will be maintaining it in liquid form. What i understand TCEP wont work. if i have to use SEC what buffer should i use. My resin is Superdex 75 prep grade and i am using 10 mM Na Acetate buffer containing 150 mM NaCl pH 4.0 for SEC.
Once again thanks for all your replies. On 3/26/10, Ho Leung Ng <[email protected]> wrote: > > Does your SDS-PAGE loading buffer contain a reducing agent like beta > mercaptoethanol? That could be responsible for the difference between > your SDS-PAGE and HPLC results. > > > ho > > On Fri, Mar 26, 2010 at 5:01 PM, CCP4BB automatic digest system > <[email protected]> wrote: > > There are 5 messages totaling 490 lines in this issue. > > > > Topics of the day: > > > > 1. TCEP effect on protein (4) > > 2. 3 PhD studentships available immediately > > > > ---------------------------------------------------------------------- > > > > Date: Thu, 25 Mar 2010 22:51:30 -0800 > > From: megha goyal <[email protected]> > > Subject: TCEP effect on protein > > > > Hi all, > > > > My protein is relatively pure except the dimer. i used 10 mM TCEP to > reduce > > it (directly added 1 M TCEP to make final volume of 10mM and kept at room > > temperature for 10 mins] then i dialysed the protein sample to remove > TCEP > > [dialysis buffer is Na acetate pH 4.0]. > > > > On performing SDS PAGE analysis after dialysis of the protein we are > getting > > no dimer band, only band of our protein is observed and same is the case > > with UV reading there is no change in it. But the HPLC analysis of the > > protein shows two peaks instead of one peak as observed before TCEP > > treatment. what can be the reason for this. Kindly guide. i need the > protein > > to formulate and conduct stability studies on the sample. the protein we > > obtain after IEX is pure except the dimer and i do not want to go for SEC > as > > it greatly reduces protein content and also is quite time consuming. any > > light on what is happening will bevery useful. > > > > thanks and regards > > > > ------------------------------ > > > > Date: Fri, 26 Mar 2010 08:37:43 +0100 > > From: Ganesh Natrajan <[email protected]> > > Subject: Re: TCEP effect on protein > > > > > > > > Hi Megha, > > > > The two peaks on the HPLC indicate that your protein is > > existing in a monomer-dimer equilibrium in solution. The dimerisation is > > most probably caused by disulphide bridges. The use of TCEP is breaking > > those disulphides and that is causing the equilibrium to move towards the > > monomeric state. However, when the TCEP is dialysed out, the disulphides > > start forming again and this is causing the equilibrum to move towards > the > > dimeric state, a process clearly hastened by the strongly oxidising pH 4 > of > > the dialysis buffer. > > > > Now it all depends on what you want to do. If you > > want to use the protein in a (largely) monomeric form, I would recommend > > that you don't dialyse out the TCEP. > > > > regards > > > > Ganesh > > > > > > ********************************************** > > Blow, blow, thou winter > > wind > > Thou art not so unkind > > As man's ingratitude; > > Thy tooth is not so > > keen, > > Because thou art not seen, > > Although thy breath be rude. > > > > -William > > Shakespeare > > ********************************************** > > > > On Thu, 25 Mar > > 2010 22:51:30 -0800, megha goyal wrote: Hi all, My protein is > > relatively pure except the dimer. i used 10 mM TCEP to reduce it > (directly > > added 1 M TCEP to make final volume of 10mM and kept at room temperature > > for 10 mins] then i dialysed the protein sample to remove TCEP [dialysis > > buffer is Na acetate pH 4.0]. On performing SDS PAGE analysis after > > dialysis of the protein we are getting no dimer band, only band of our > > protein is observed and same is the case with UV reading there is no > change > > in it. But the HPLC analysis of the protein shows two peaks instead of > one > > peak as observed before TCEP treatment. what can be the reason for this. > > Kindly guide. i need the protein to formulate and conduct stability > studies > > on the sample. the protein we obtain after IEX is pure except the dimer > and > > i do not want to go for SEC as it greatly reduces protein content and > also > > is quite time consuming. any light on what is happening will bevery > useful. > > thanks and regards > > > > -- > > > > ------------------------------ > > > > Date: Fri, 26 Mar 2010 08:49:55 +0100 > > From: Matthias Zebisch <[email protected]> > > Subject: Re: TCEP effect on protein > > > > Hi Ganesh and Mega! > > > > I do not agree with Ganesh. I assume, Megha, that truly reversed > > phaseHPLC was used. This is a denaturing method and the natural > > disulfide should not form again during the run. > > Also pH 4 can not be described "oxidizing". Actually, reduced proteins > > are often dialyzed against acidic buffers to prevent disulfide formation > > via the thiolate anion. > > Still, a reducing agent may be used during the run? > > > > Sorry that I can not offer a solution to the real problem. More > > experimental details may be necessary. > > > > Bets regards, Matthias > > > > Am 3/26/2010 8:37 AM, schrieb Ganesh Natrajan: > >> > >> Hi Megha, > >> > >> The two peaks on the HPLC indicate that your protein is existing in a > >> monomer-dimer equilibrium in solution. The dimerisation is most > >> probably caused by disulphide bridges. The use of TCEP is breaking > >> those disulphides and that is causing the equilibrium to move towards > >> the monomeric state. However, when the TCEP is dialysed out, the > >> disulphides start forming again and this is causing the equilibrum to > >> move towards the dimeric state, a process clearly hastened by the > >> strongly oxidising pH 4 of the dialysis buffer. > >> > >> Now it all depends on what you want to do. If you want to use the > >> protein in a (largely) monomeric form, I would recommend that you > >> don't dialyse out the TCEP. > >> > >> regards > >> > >> Ganesh > >> > >> ********************************************** > >> Blow, blow, thou winter wind > >> Thou art not so unkind > >> As man's ingratitude; > >> Thy tooth is not so keen, > >> Because thou art not seen, > >> Although thy breath be rude. > >> > >> -William Shakespeare > >> ********************************************** > >> > >> On Thu, 25 Mar 2010 22:51:30 -0800, megha goyal <[email protected]> > >> wrote: > >> > >> Hi all, > >> My protein is relatively pure except the dimer. i used 10 mM TCEP > >> to reduce it (directly added 1 M TCEP to make final volume of 10mM > >> and kept at room temperature for 10 mins] then i dialysed the > >> protein sample to remove TCEP [dialysis buffer is Na acetate pH > 4.0]. > >> On performing SDS PAGE analysis after dialysis of the protein we > >> are getting no dimer band, only band of our protein is observed > >> and same is the case with UV reading there is no change in it. But > >> the HPLC analysis of the protein shows two peaks instead of one > >> peak as observed before TCEP treatment. what can be the reason for > >> this. Kindly guide. i need the protein to formulate and conduct > >> stability studies on the sample. the protein we obtain after IEX > >> is pure except the dimer and i do not want to go for SEC as it > >> greatly reduces protein content and also is quite time consuming. > >> any light on what is happening will bevery useful. > >> thanks and regards > >> > >> -- > >> > > > > > > -- > > **************************************************** > > Dr. Matthias Zebisch > > Universität Leipzig > > Biotechnologisch-Biomedizinisches Zentrum > > Strukturanalytik von Biopolymeren > > Deutscher Platz 5 > > 04103 Leipzig > > Germany > > Phone: 0049-341-97-31323 (lab) -31312 (office) > > Fax : 0049-341-97-31319 > > email: [email protected] > > **************************************************** > > > > ------------------------------ > > > > Date: Fri, 26 Mar 2010 10:53:07 +0000 > > From: "Hough, Mike" <[email protected]> > > Subject: 3 PhD studentships available immediately > > > > Dear CCP4bb, > > > > The following three studentship positions are available immediately at > the University of Liverpool. Could you please pass these details on to any > students in your departments or elsewhere who may be interested? Replies to > the email addresses listed below, rather than to me please. > > > > Thanks, > > > > Mike > > > > > > School of Biological Sciences, University of Liverpool > > > > 4-year joint BBSRC PhD Studentships – applications invited immediately > > > > Three PhD studentships are available in the Molecular Biophysics Group > for research in the area of x-ray structural biology using the most advanced > synchrotron radiation sources and the new light sources in the form of x-ray > free electron lasers (XFELs). The studentships involve collaborations with > scientists at the DIAMOND Light Source, UK and at the RIKEN Spring-8 Center, > Japan. The successful candidates will have the exciting opportunity of > spending up to 2 years at these premier science facilities working at the > leading edge of structural biology. We are looking for applications now from > highly committed and motivated graduates from any of the natural sciences > who are ordinarily resident in the UK. Contact details and links to detailed > project descriptions are given below: > > > > “Long wavelength X-ray diffraction experiments for structural studies > from biological redox systems” > > [Strange, UoL & Wagner, DIAMOND] > > see: http://www.biophysics.liv.ac.uk/PhD_DLS.pdf > > contact [email protected] > > > > “Metalloproteomics: Structure and function studies of metalloproteins of > biomedical importance” > > [Hasnain, UoL & Shiro, RIKEN] > > see: http://www.biophysics.liv.ac.uk/PhD_RIKEN_Metalloprotein.pdf > > contact: [email protected] > > > > “New Science exploration from XFEL: A new paradigm for structural > visualisation of macromolecules” > > [Grossmann, UoL & Ishikawa, RIKEN] > > see: http://www.biophysics.liv.ac.uk/PhD_RIKEN_XFEL.pdf > > contact: [email protected] > > > > ------------------------------ > > > > Date: Fri, 26 Mar 2010 09:40:48 -0700 > > From: James Holton <[email protected]> > > Subject: Re: TCEP effect on protein > > > > Yes, it is possible that the disulfide is just re-forming when the TCEP > > is gone. pH 4 is not favorable for the oxidation, but does not prohibit > > it either. Especially if there are traces of metal ions around. I > > learned this the hard way, as trace metals can catalyze the oxidation of > > methionine and selenomethionine as well. In my hands, adding a pinch of > > EDTA (~1 mM final conc.) to the fraction coming off the HPLC stabilized > > the reduced species. Since the HPLC I was using at the time was made of > > metal (and showed signs of rust around some of the fittings), it is > > perhaps not surprising that I had a few ppm of Fe++ in the mobile phase. > > > > -James Holton > > MAD Scientist > > > > megha goyal wrote: > >> Hi all, > >> > >> My protein is relatively pure except the dimer. i used 10 mM TCEP to > >> reduce it (directly added 1 M TCEP to make final volume of 10mM and > >> kept at room temperature for 10 mins] then i dialysed the protein > >> sample to remove TCEP [dialysis buffer is Na acetate pH 4.0]. > >> > >> On performing SDS PAGE analysis after dialysis of the protein we are > >> getting no dimer band, only band of our protein is observed and same > >> is the case with UV reading there is no change in it. But the HPLC > >> analysis of the protein shows two peaks instead of one peak as > >> observed before TCEP treatment. what can be the reason for this. > >> Kindly guide. i need the protein to formulate and conduct stability > >> studies on the sample. the protein we obtain after IEX is pure except > >> the dimer and i do not want to go for SEC as it greatly reduces > >> protein content and also is quite time consuming. any light on what is > >> happening will bevery useful. > >> > >> thanks and regards > > > > ------------------------------ > > > > End of CCP4BB Digest - 25 Mar 2010 to 26 Mar 2010 (#2010-82) > > ************************************************************ > > >
