ISSN:
1615-6102
Schlagwort(e):
Immunofluorescence staining
;
Higher plant tubulin
;
Microtubules
;
Monoclonal antibodies
;
Mung bean
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
Notizen:
Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two α-tubulin subunits, MBT2 and MBT3, of which MBT3 is a minor component, and one Β-tubulin, MBT1. Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution. The properties of monoclonal antibody (K4E7G3) were examined by Western blot analysis and indirect immunofluorescence studies. K4E7G3 reacts with MBT2 and MBT3 α-tubulin subunits of mung bean tubulin, but not with MBT1 Β-tubulin nor with the α- and Β-subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K4E7G3 and with other monoclonal antibodies that are known to be specific to the α-subunit of yeast tubulin and α- or Β-subunit of mammalian brain tubulin. MBT2 and MBT3 are shown to be similar but not identical and are quite different from MBT1 and the Β-subunit of sheep brain tubulin. K4E7G3 reacts with peptide fragments in MBT2 and MBT3 that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain α-tubulin. It is concluded that K4E7G3 and another monoclonal antibody, K2D7B8, which has similar properties, are relatively specific for plant α-tubulin. In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K4E7G3 and K2D7B8 do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K4E7G3 to react with brain tubulin subunits on Western blot analysis.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF01279907
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