Ni-affinity purification and reducing agents

[U12201 at uicvm.uic.edu]

You could send the whole thing through a column of immobilized
DTT - Pierce sells one called :  ReduceImm - that way you
would get the reduction without contamination of DTT.

In article <409vtv$88b at biovax.biobase.dk>, Per Mygind <perm> says:
>
>-- Fellow Readers
>
>        My question to the readers is concerning affinity-purification of     binant
>recom
>proteins, by use of Ni-NTA columns.
>        I've constructed and purified recombinant proteins of different kinds,but
>I've run my head against a tedious problem. So far, I've used
>beta-mercaptoethanol as a reducing agent, if the fusionprotein in question was
>containing cysteine-residues. One drawback by using this reagent, is that it
>binds covalently to the residues and thereby manipulating an antigenic
>property
>of the fusionprotein.
>        My question is, can you use a reducing agent like DTT or DTE instead
>of
>beta-mercaptoethanol. I've heard that it is not possible with Ni-NTA columns,
>have anyone got experience with that ?
>Is it maybe possible to remove beta-mercaptoethanol upon purification ?
>
>Any comments or suggestions will be greatly appreciated
>
>Regards
>
>          Per Mygind , Medical Microbiology , University of Aarhus , Denmark
>******************************************************************************
>
>MY OWN PERSONAL FINGERTOUCH
>
>******************************************************************************
>
>> -- Fellow Readers
>>
>>         My question to the readers is concerning affinity-purification of   o
>rec
>> proteins, by use of Ni-NTA columns.
>>         I've constructed and purified recombinant proteins of different     ,
>kinds
>> I've run my head against a tedious problem. So far, I've used
>> beta-mercaptoethanol as a reducing agent, if the fusionprotein in question  s
>wa
>> containing cysteine-residues. One drawback by using this reagent, is that it
>> binds covalently to the residues and thereby manipulating an antigenic      t
>proper
>> of the fusionprotein.
>>         My question is, can you use a reducing agent like DTT or DTE insteado
>> beta-mercaptoethanol. I've heard that it is not possible with Ni-NTA
>columns,
>> have anyone got experience with that ?
>> Is it maybe possible to remove beta-mercaptoethanol upon purification ?
>>
>> Any comments or suggestions will be greatly appreciated
>>
>> Regards
>>
>>           Per Mygind , Medical Microbiology , University of Aarhus , Denmark
>>                                                                             *
>*****************************************************************************
>>
>> MY OWN PERSONAL FINGERTOUCH
>>
>>                                                                             *
>*****************************************************************************
>>