--------------21AB64FA4CC9
Content-Type: text/plain; charset=us-ascii
Content-Transfer-Encoding: 7bit
-- Gottfried Proess Tel.: + 32 4 366 01 50 Fax : + 32 4 365 16 04 E-Mail: g.proess@eurogentec.com Web-Site : http://www.eurogentec.com <==== visit it !--------------21AB64FA4CC9 Content-Type: message/rfc822 Content-Transfer-Encoding: 7bit Content-Disposition: inline
Message-ID: <37A1C212.2094@eurogentec.com> Date: Fri, 30 Jul 1999 08:17:38 -0700 From: Gottfried Proess <g.proess@eurogentec.com> Reply-To: g.proess@eurogentec.com Organization: Eurogentec Bel S.A. X-Mailer: Mozilla 3.0 (Win16; I) MIME-Version: 1.0 To: Latif Kazim <kazim@sc3101.med.buffalo.edu> Subject: Re: Antipeptide antibodies References: <199907271716.NAA02114@post.aecom.yu.edu> <199907291821.UAA05607@mail.eurogentec.be> Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit
Latif Kazim wrote: > > Dear Gottfried: > > Thank you for your response. Yes, you are correct, and I apologize > for any ambiguity in the message I sent to the bulletin board. As far > as the his-tag peptides are concerned (the reference is Biotechniques > (1998) 25:29-32), 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 > agarose? > > Latif Kazim > > >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. > >> > >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. > >-- > > ---------------------------------------- > 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,
thank you for your reply. We did not compare the titers between the different methods. But to my opinion, the titers should not be higher than with the MAP method. Normally, agarose is not immunogenic to enhance antibody response, effect that you get with the KLH-coupled peptide. The advantage of the MAP peptide is that there is, in comparison to KLH-coupled peptide, more peptide and no other peptide structures (KLH) available for antibody production, resulting generally in quite good titers of anti-MAP peptide antibodies, but which in our hands work less often against the protein than with the KLH method.
Neither of these advantages can be supposed with the agarose method to my opinion, one has not the advantage of boosting immmune answer by the immunogenic KLH and one has not the advantage of immunizing with only the interesting peptide present in large quantity. But these are suppositions and have not been verified in our labs.
I'll take a look at the mentioned article.
Best regards
-- Gottfried Proess Tel.: + 32 4 366 01 50 Fax : + 32 4 365 16 04 E-Mail: g.proess@eurogentec.com Web-Site : http://www.eurogentec.com <==== visit it !--------------21AB64FA4CC9--