Phaseolus vulgaris agglutinin is the name ascribed to a family of lectins, each of which consists of four subunits. There are two different types of subunits. One appears to be involved primarily in red cell agglutination and has been designated the “E” subunit (for erythroagglutinin). The other type is involved in lymphocyte agglutination and mitogenic activity and has been termed the “L” subunit (for leucoagglutinin). These subunits combine to produce five isolectins.
The unconjugated PHA-L has been found to be an excellent, specific marker for use in anterograde neuronal tracing.
Reconstitution: The lectin should be reconstituted 10 mM phosphate, pH 8.0. If reconstituted in 1 ml of 10 mM phosphate, the resulting solution will be 5 mM phosphate, 10 mM NaCl with trace CaCl2. After reconstitution, and if appropriate for use, a preservative such as sodium azide (0.04%) may be added. If the lectin is to be used for mitogenic assays, filter solution through a sterile 0.22 µm filter immediately after reconstitution, aliquot and store frozen.
NOTE: Mitogenic activity may be diminished by repeated freezing and thawing.
Anterograde Neuronal Tracing: For neuronal transport studies, reconstitute in 0.2 ml of 10 mM sodium phosphate, pH 8.0.Both the pH and ionic strength of the reconstituting buffer are important for optimal anterograde transport. A specific protocol for this application is available on request.
PHA-L, with four L type subunits, does not agglutinate red cells but is a potent mitogen. The other three isolectins, designated E3L1, E2L2, and E1L3, have erythroagglutinating and mitogenic activities proportional to the number of respective E or L subunits. We have termed the mixture of the five isolectins PHA (E+L).
After iontophoretic injection of unconjugated PHA-L, the approximate rate of anterograde transport is about 4-6 mm/day, with survival periods of over 18 days having been observed. Once transported, the unconjugated PHA-L is best visualized for light microscopy with antibody to the lectin, followed by the appropriate detection system and substrate. Fluorescent staining can also be used for a faster staining procedure. A complete applications protocol is available upon request.