Screening a Library by Immunological Procedures

Instead of looking for DNA/DNA hybrids to identify the gene of interest from a library, the protein itself can be identified by immunological screening. This method relies on the production of the protein encoded by the gene of interest and therefore assumes that the cloned gene is efficiently expressed under the experimental conditions. That is, each of the library inserts must have transcriptional and translational start sequences as well as stop sequences. Usually, the library vector supplies these sequences, since the promoters from the genomic DNA will not usually be cloned still attached to the genes they control (see below). When the protein is expressed, it is detected by binding to an antibody. This means that antibody to the encoded protein (or a closely related protein from another organism) must be available.

In order to screen an expression library, the bacteria expressing the library inserts are grown on master plates and samples of each bacterial colony are transferred to a suitable membrane. The cells are lysed and the released proteins are attached to the membrane. The membrane is then treated with a solution of the appropriate antibody. After excess primary antibody is washed away, a second antibody that is specific for the primary antibody is added.This will bind any primary antibody it encounters (Fig. 22.25). This secondary antibody carries the detection system, such as alkaline phosphatase, which converts a colorless substrate, such as X-phos, to a colored product (see Ch. 21). If X-phos is used, the region on the membrane where the secondary antibody is bound turns blue. The blue spots must be aligned with the original bacterial colonies. The DNA from the bacteria containing the insert encoding the protein of interest can then be isolated.

The reason for using two different antibodies is to allow flexibility and amplify the signal. Antibodies to the protein of interest are made by injecting a rabbit with the antibody Protein made by the immune system to recognize and bind to foreign proteins or other macromolecules immunological screening Screening procedure that relies on the specific binding of antibodies to the target protein target DNA DNA that is the target for binding by a probe during hybridization or the target for amplification by PCR

Bacterial colonies on agar each carry a cloned fragment of DNA

tRANSFER TO MEMBRANE OR FILTER

Bacterial colonies on agar each carry a cloned fragment of DNA

tRANSFER TO MEMBRANE OR FILTER

Lysis of bacterial cells

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