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) > ************************************************************ >
