Refine
Has Fulltext
- yes (3)
Is part of the Bibliography
- yes (3)
Document Type
- Journal article (1)
- Book (1)
- Doctoral Thesis (1)
Language
- English (3) (remove)
Keywords
- monoclonal antibodies (3)
- Drosophila melanogaster (2)
- Monoklonaler Antikörper (2)
- Proteine (2)
- Synapse (2)
- Taufliege (2)
- monoklonale Antikörper (2)
- synaptic proteins (2)
- synaptische Proteine (2)
- cell staining (1)
For a large fraction of the proteins expressed in the human brain only the primary
structure is known from the genome project. Proteins conserved in evolution can
be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal
antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and
characterized with the aim to identify the target antigen. The mAb ab52 was found
to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western
blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a
single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15
(epidermal growth factor receptor pathway substrate clone 15) to be a strong
candidate. Another mAb from the library, aa2, was already found to recognize
EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and
2D electrophoretic separations revealed similar patterns, hence indicating that both
antigens could represent the same protein. Finally absence of the wild-type signal
in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52
antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila
homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for
applications like immunoprecipitation (IP). It has already been submitted to the
Developmental Studies Hybridoma Bank (DSHB) to be easily available for the
entire research community.
The mAb na21 was also found to be an IgM. It recognizes a membrane associated
antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature
of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of
the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to
biochemically purify the endogenously expressed protein from the tissue, gave
99
promising results but could not be completed due to lack of time. Thus
biochemical purification of the protein seems possible in order to facilitate its
identification by mass spectrometry. Several other mAbs were studied for their
staining pattern on cryosections and whole mounts of Drosophila brains. However,
many of these mAbs stained very few structures in the brain, which indicated that
only a very limited amount of protein would be available as starting material.
Because these antibodies did not produce signals on Western blots, which made it
impossible to enrich the antigens by electrophoretic methods, we did not attempt
their purification. However, the specific localization of these proteins makes them
highly interesting and calls for their further characterization, as they may play a
highly specialized role in the development and/or function of the neural circuits
they are present in. The purification and identification of such low expression
proteins would need novel methods of enrichment of the stained structures.
Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.
For a large fraction of the proteins expressed in the human brain only the primary structure is known from the genome project. Proteins conserved in evolution can be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and characterized with the aim to identify the target antigen. The mAb ab52 was found to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15 (epidermal growth factor receptor pathway substrate clone 15) to be a strong candidate. Another mAb from the library, aa2, was already found to recognize EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and 2D electrophoretic separations revealed similar patterns, hence indicating that both antigens could represent the same protein. Finally absence of the wild-type signal in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52 antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for applications like immunoprecipitation (IP). It has already been submitted to the Developmental Studies Hybridoma Bank (DSHB) to be easily available for the entire research community. The mAb na21 was also found to be an IgM. It recognizes a membrane associated antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to biochemically purify the endogenously expressed protein from the tissue, gave promising results but could not be completed due to lack of time. Thus biochemical purification of the protein seems possible in order to facilitate its identification by mass spectrometry. Several other mAbs were studied for their staining pattern on cryosections and whole mounts of Drosophila brains. However, many of these mAbs stained very few structures in the brain, which indicated that only a very limited amount of protein would be available as starting material. Because these antibodies did not produce signals on Western blots, which made it impossible to enrich the antigens by electrophoretic methods, we did not attempt their purification. However, the specific localization of these proteins makes them highly interesting and calls for their further characterization, as they may play a highly specialized role in the development and/or function of the neural circuits they are present in. The purification and identification of such low expression proteins would need novel methods of enrichment of the stained structures.