Advanced Concepts

The Western blot method is used to confirm enzyme-linked immunoassay results for human immunodeficiency virus (HIV) and hepatitis C virus among other organisms. In this procedure, known HIV proteins are separated by electrophoresis and transferred and bound to a nitrocellulose membrane. The patient's serum is overlaid on the membrane, and antibodies with specificity to HIV proteins bind to their corresponding protein. Unbound patient antibodies are washed off, and binding of antibodies is detected by adding a labeled antihuman immunoglobulin antibody. If HIV antibodies are present in the patient's serum, they can be detected with antihuman antibody probes appearing as a dark band on the blot corresponding to the specific HIV protein to which the antibody is specific.

Probes

The probe for Southern and Northern blots is a single-stranded fragment of nucleic acid. The purpose of the probe is to identify one or more sequences of interest within a large amount of nucleic acid. The probe therefore should hybridize specifically with the target DNA or RNA that is to be analyzed. The probe can be RNA, denatured DNA, or other modified nucleic acids. Peptide nucleic acids (PNAs) and locked nucleic acids have also been used as probes. These structures contain normal nitrogen bases that can hybridize with complementary DNA or RNA, but the bases are connected by backbones different from the natural phosphodiester backbone of DNA and RNA. These modified backbones are resistant to nuclease degradation and, because of a reduced negative charge on their backbone, can hybridize more readily to target DNA or RNA.

Probes for Western blots are specific binding proteins or antibodies. A labeled secondary antibody directed against the primary binding protein is then used for the visualization of the protein band of interest.

Was this article helpful?

0 0

Post a comment