I am interested in preparing a phagemid library. I would like to know if it is very expensive (more or less) and if it needs lots of lab experience. I would be grateful for any answer!
Check out this protocol:
Schaefer, J. V., Honegger, A., and Plückthun, A. (2010) in Antibody Engineering: Construction of scFv fragments from hybridoma or spleen cells by PCR assembly (Kontermann, R., and Dübel, S., eds) Vol. Vol. 1, 2nd edit., pp. 21-44, Springer-Verlag, Berlin Heidelberg, Germany.
http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-30700
it is an updated and improved version to this:
Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., and Plückthun, A. (1997). Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35-55.
http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-11300
The protocols start from spleen cells rather than blood
Of course, if you plan to start from a different species than mouse, the primer sets need to be adapted.
phage display library wil be technically and financially less consuming.
I hope this helps!
Marcelo
Check out this protocol:
Schaefer, J. V., Honegger, A., and Plückthun, A. (2010) in Antibody Engineering: Construction of scFv fragments from hybridoma or spleen cells by PCR assembly (Kontermann, R., and Dübel, S., eds) Vol. Vol. 1, 2nd edit., pp. 21-44, Springer-Verlag, Berlin Heidelberg, Germany.
http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-30700
it is an updated and improved version to this:
Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., and Plückthun, A. (1997). Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35-55.
http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-11300
The protocols start from spleen cells rather than blood
Of course, if you plan to start from a different species than mouse, the primer sets need to be adapted.
Generating a phagemid library is not a very costly affair, if your lab has basic molecular biology facilities like RNA extraction kit, cDNA synthesis kits, phagemid, primers, thermocycler, appropriate restriction enzymes, electrocompetent cells, etc. However, the troublesome part is selection of specific antibodies for your antigen from this library. I think people will be able to help you much better if you can explain a bit about what you plan to do.
Most common protocols are available. I guess the basic know how would be molecular techniques. Just keep in mind depending on the type of library you wish to make, the diversity is important. With that said, cloning a library would pose some challenges compared to single clone experiments.
we constructed camel/mouse/rabbit/chicken immune library for selecting antibody of high affiity. you may contact me by email [[email protected]]. then I will give you a price depends on your request.
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