Re: [ccp4bb] TCEP effect on protein
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 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 > 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 > > 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 > > 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 > > 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
Re: [ccp4bb] TCEP effect on protein
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 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 > 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 > 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 > 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
Re: [ccp4bb] 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
Re: [ccp4bb] 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 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: matthias.zebi...@bbz.uni-leipzig.de
Re: [ccp4bb] 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 --
[ccp4bb] 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
