Nucleotide antibody specificity

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

An EMSAs is a simple assay to determine if a DNA sequence is able to bind a specific protein. Purified, cellular, or nuclear protein extract is incubated with labeled double-stranded oligonucleotide DNA probes, usually 20-25 nucleotides long. Each probe contains the SNP nucleotide (one per probe) and the identical sequence corresponding to the sequence around the SNP. The mixture is then run on a nondenaturing gel. If the DNA sequence binds the protein, there is a shift in the mobility of the complex (e.g., lane 2, Fig. 12.3). Specific and nonspecific competitors are often added to demonstrate specificity of the shift (lanes 5-7, Fig. 12.3). If an antibody... 

Proteins are the most abundant of cellular components. They include enzymes, antibodies, hormones, transport molecules, and even components for the cytoskeleton of the cell itself. Proteins are also informational macromolecules, the ultimate heirs of the genetic information encoded in the sequence of nucleotide bases within the chromosomes. Structurally and functionally, they are the most diverse and dynamic of molecules and play key roles in nearly every biological process. Proteins are complex macromolecules with exquisite specificity each is a specialized player in the orchestrated activity of the cell. Together they tear down... 

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

Surface-exposed parts of the protein have been distinguished by their ability to bind specific antibodies and proteolytic enzymes under nondenaturing conditions (see Figure 16). Many of these epitopes are located at positions in the sequence that correspond to the external side of the suggested nucleotide-binding fold (Mate et al., 1992). Antibodies to fluorescein bind only to denatured FITC-labeled Ca2+-ATPase, and not to the native FITC-labeled enzyme. This is consistent with a location of bound FITC in a hydrophobic cleft corresponding to the ATP site. 

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