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
<[log in to unmask]> 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 <[log in to unmask]>
> 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 <[log in to unmask]>
> 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 <[log in to unmask]>
> 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 <[log in to unmask]>
>> 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: [log in to unmask]
> ****************************************************
>
> ------------------------------
>
> Date: Fri, 26 Mar 2010 10:53:07 +0000
> From: "Hough, Mike" <[log in to unmask]>
> 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 [log in to unmask]
>
> “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: [log in to unmask]
>
> “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: [log in to unmask]
>
> ------------------------------
>
> Date: Fri, 26 Mar 2010 09:40:48 -0700
> From: James Holton <[log in to unmask]>
> 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)
> ************************************************************
>
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