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Chapter: Genetics and Molecular Biology: Advanced Genetic Engineering

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Finding Clones Using Antibodies Against a Protein

Cloning a gene becomes easier than described above if sufficient quan-tities of its gene product are available to permit raising antibodies against the protein.

Finding Clones Using Antibodies Against a Protein

Cloning a gene becomes easier than described above if sufficient quantities of its gene product are available to permit raising antibodies against the protein. DNA from the organism is cloned into a vector designed to provide for transcription and translation of the inserted DNA. The DNA is best cloned into a site transcribed from a controllable upstream promoter and also containing an upstream ribosome binding site and protein translation initiation sequence as well (Fig. 10.2). A fragment of DNA inserted into the site and containing an open reading frame is translated if it is fused in frame with the initiation sequence. As in screening with oligonucleotides, a replica plate is made, cells on the plate are grown, and the controllable promoter is induced. The cells are lysed, the proteins are immobilized on a filter, antibody is added,



Figure 10.2 Structure of a vector suitable for antibody screening for theinsertion of open reading frames of a specific protein.

and then areas with bound antibody are revealed as described below. The colony from the corresponding position on the replica plate can then be picked and studied.

 

Molecules of one particular antibody type bind to just one particular shape found in some other macromolecule. This is defined as their antigen. Almost any protein can be used as an antigen to elicit the synthesis of antibodies. Thus antibodies provide highly selective agents for the detection of specific proteins. The antibody selectivity for bind-ing to the correct shape compared to binding to incorrect shapes is roughly the same as the hybridization selectivity of nucleic acids.

 

Although radioactive antibody could be used to detect antigen syn-thesized by candidate clones, it is not efficient, for different antibodies would then have to be made radioactive for the detection of different proteins. The A protein from Staphylococcus aureus provides a more general detection method. This protein binds to a portion of the anti-body molecule so that one sample of radioactive or enzymatically tagged Staphylococcus aureus A protein suffices for the detection of manydifferent antibody-protein complexes (Fig. 10.3). Another detection method is to use antibodies themselves as labels. The protein on the filter paper can be incubated with antibodies specific for the protein that were raised in mice. Then rabbit antibodies that have been raised against mouse antibodies can be added. These will detect and bind to most mouse antibodies. The enzyme alkaline phosphatase can be linked


Figure 10.3 The use of radioactiveStaphylococcusA protein to identify anti-body-antigen complexes in Western transfers.



to rabbit-antimouse antibodies (Fig. 10.4). Their location can be marked by adding colorless substrate for alkaline phosphatase whose hydrolysis product is highly colored and insoluble. The product shows the location of protein to which the mouse antibody bound, to which, in turn, the rabbit antibodies containing alkaline phosphatase bound.

 

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