By Nina Strebe, Frank Breitling, Dieter Moosmayer, Bodo Brocks, Stefan Dübel (auth.), Roland Kontermann, Stefan Dübel (eds.)
Antibodies are integral instruments for examine, analysis, and remedy. Recombinant ways let the amendment and development of approximately all antibody homes, equivalent to affinity, valency, specificity, balance, serum half-life, effector capabilities, and immunogenicity.
Antibody Engineering offers a complete toolbox masking the well-established fundamentals but in addition many fascinating new innovations. The protocols mirror the newest "hands on" wisdom of key laboratories during this nonetheless fast-moving box. rookies will enjoy the confirmed step by step protocols, which come with worthy sensible recommendation; skilled antibody engineers will delight in the hot rules and techniques. The ebook is a useful source for all these engaged in antibody learn and improvement.
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Extra info for Antibody Engineering
The most critical steps were already highlighted directly following the instructions in the different subsections. (a) In case of low transformation yields, check whether the problem is the transformation itself or rather the ligation. To investigate the quality of ligation, analyzing an aliquot by agarose gel electrophoresis might indicate any problems caused by nucleases. Furthermore, it might be informative to compare the ligation efficiency of SfiI digested PCR product with inserts derived from plasmid digestion.
We also tried to ensure similar annealing temperatures with the different genes, as well as keeping the degeneracy on the DNA level as small as possible. Furthermore, we avoided pronounced secondary structures within the oligonucleotides such as hairpin loops or primer-dimers (which were checked against themselves using the appropriate analysis tools in the Vector NTI software (Invitrogen)). The primers shown below are the result of this iterative process and have also been tested with a slightly different overhang.
Coli alkaline phosphatase (AP) gene can be found in Shuttleworth et al. (1986). For the EcoRI/HindIII cloning cassette the two internal EcoRI sites of the AP gene have been removed by silent mutations. V. Schaefer et al. Library Preparation/Construction 1. For desalting the DNA prior to electroporation, apply the ligation mix to StrataClean Resin (Stratagene; hydroxylated silica, binding proteins with a high affinity, while having a low affinity for DNA at near neutral pH), followed by precipitation in 70% ethanol.
Antibody Engineering by Nina Strebe, Frank Breitling, Dieter Moosmayer, Bodo Brocks, Stefan Dübel (auth.), Roland Kontermann, Stefan Dübel (eds.)