Dear Gottfried:
Yes, you are correct, and I apologize for any ambiguity in the message I sent
to the bulletin board. Regarding his-tagged peptides, the reference
mentioned
by GSarath is Biotechniques (1998) 25:29-32, and it's also discussed in
QiagenNews 3: 3-6(1999). I agree with your comments that they are more costly
and perhaps more difficult to synthesize, and that the KLH method is easy and
works well - we routinely make MAP and KLH-conjugated peptides for
immunization, but we have never tried agarose-conjugated peptides or proteins.
Perhaps, the question should be : has anyone compared the titers and
specificities of antisera raised by immunizing with peptides prepared as MAPs,
KLH (or other protein) conjugates, and linked (by any method) to agarose?
Also, in the references to the his-tag method, mention is made that 6XHis is
poorly immunogenic in most species. I wonder how consistent and well
documented this is?
Latif Kazim
At 12:40 PM 7/29/99 -0700:
>Latif Kazim wrote:
>>
>> Dear Colleagues:
>>
>> One of our researchers recently asked the following question about a method
>> for making antipeptide antisera:
>>
>> >Do you have knowledge/experience with direct use of synthetic
>> >peptides for producing antisera--particularly the 6xhis-tagged peptide
>> >method?? Apparently the peptide is bound to Ni-NTA agarose and used
>> >directly.
>>
>> Any responses would be appreciated.
>>
>> Thanks,
>>
>> Latif Kazim
>>
>> ----------------------------------------
>> A. Latif Kazim, Ph.D.
>> Biopolymer Facility
>> Science-4
>> Roswell Park Cancer Institute
>> Elm & Carlton Sts.
>> Buffalo, NY 14263
>>
>> E-mail: kazim@sc3101.med.buffalo.edu
>>
>> Phone: (716) 845-8923
>> Fax: (716) 845-7621
>
>
>
>Dear Latif,
>
>having read the answer of <gsarath@unlnotes.unl.edu>, there is maybe a
>misunderstanding somewhere. What I understood from your question is that
>the peptide is synthesized with a 6His-tag, bound to Ni-NTA agarose and
>injected like this for antibody production. Am I right or does your
>question effectively concern the antiserum purification on a Ni-NTA
>column on which the 6His-tag peptide has been fixed?
>
>If I am correct, I cannot see any real advantage in doing this type of
>antiserum production. The addition of 6 histidines makes the peptide
>more expensive and maybe even more difficult to synthesize. The classic
>method of coupling part of the untagged peptide to KLH works very fine
>for antibody production and the remaining uncoupled peptide can be used
>easily for subsequent antiserum purification on an affinity column using
>again the same type of coupling reaction to an adapted matrix.
>
>But if you want to try antibody production using the agarose beads, I
>would not suspect any difficulties or problems in doing this. We did
>several successful antibody productions with agarose-bound proteins, but
>not yet with peptides.
>
>I hope this helps a little.
>--
>Gottfried Proess
>Tel.: + 32 4 366 01 50 Fax : + 32 4 365 16 04
>E-Mail: g.proess@eurogentec.com
>
----------------------------------------
A. Latif Kazim, Ph.D.
Biopolymer Facility
Science-4
Roswell Park Cancer Institute
Elm & Carlton Sts.
Buffalo, NY 14263
E-mail: kazim@sc3101.med.buffalo.edu
Phone: (716) 845-8923
Fax: (716) 845-7621
--=====================_13083935==_.ALT
Content-Type: text/html; charset="us-ascii"
Dear Gottfried:
--=====================_13083935==_.ALT--
Yes, you are correct, and I apologize for any ambiguity in the message I
sent to the bulletin board. Regarding his-tagged peptides,
the reference mentioned by GSarath is Biotechniques (1998) 25:29-32, and
it's also discussed in QiagenNews 3: 3-6(1999).
I agree with your comments that they are more costly and perhaps
more difficult to synthesize, and that the KLH method is easy and works
well - we routinely make MAP and KLH-conjugated peptides for
immunization, but we have never tried agarose-conjugated peptides or
proteins. Perhaps, the question should be : has anyone compared the
titers and specificities of antisera raised by immunizing with peptides
prepared as MAPs, KLH (or other protein) conjugates, and linked (by
any method) to agarose? Also, in the references to the his-tag
method, mention is made that 6XHis is poorly immunogenic in most
species. I wonder how consistent and well documented this is?
Latif Kazim
At 12:40 PM 7/29/99 -0700:
>Latif Kazim wrote:
>>
>> Dear Colleagues:
>>
>> One of our researchers recently asked the following question
about a method
>> for making antipeptide antisera:
>>
>> >Do you have knowledge/experience with direct use of
synthetic
>> >peptides for producing antisera--particularly the
6xhis-tagged peptide
>> >method?? Apparently the peptide is bound to Ni-NTA
agarose and used
>> >directly.
>>
>> Any responses would be appreciated.
>>
>> Thanks,
>>
>> Latif Kazim
>>
>> ----------------------------------------
>> A. Latif Kazim, Ph.D.
>> Biopolymer Facility
>> Science-4
>> Roswell Park Cancer Institute
>> Elm & Carlton Sts.
>> Buffalo, NY 14263
>>
>> E-mail: kazim@sc3101.med.buffalo.edu
>>
>> Phone: (716) 845-8923
>> Fax: (716) 845-7621
>
>
>
>Dear Latif,
>
>having read the answer of <gsarath@unlnotes.unl.edu>, there is
maybe a
>misunderstanding somewhere. What I understood from your question is
that
>the peptide is synthesized with a 6His-tag, bound to Ni-NTA agarose
and
>injected like this for antibody production. Am I right or does
your
>question effectively concern the antiserum purification on a
Ni-NTA
>column on which the 6His-tag peptide has been fixed?
>
>If I am correct, I cannot see any real advantage in doing this type
of
>antiserum production. The addition of 6 histidines makes the
peptide
>more expensive and maybe even more difficult to synthesize. The
classic
>method of coupling part of the untagged peptide to KLH works very
fine
>for antibody production and the remaining uncoupled peptide can be
used
>easily for subsequent antiserum purification on an affinity column
using
>again the same type of coupling reaction to an adapted matrix.
>
>But if you want to try antibody production using the agarose beads,
I
>would not suspect any difficulties or problems in doing this. We
did
>several successful antibody productions with agarose-bound proteins,
but
>not yet with peptides.
>
>I hope this helps a little.
>--
>Gottfried Proess
>Tel.: + 32 4 366 01 50 Fax : + 32 4 365 16 04
>E-Mail: g.proess@eurogentec.com
>