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Monoclonal antibody HFD9 identifies a novel 28 kDa integral membrane protein on the cis-Golgi
V N Subramaniam1, J Krijnse-Locker, B L Tang
1Membrane Biology Laboratory, National University of Singapore.
Abstract:
We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly-conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers.
Insights
Researchers developed a monoclonal antibody (HFD9) identifying a novel 28 kDa integral membrane protein (p28) on the cis-Golgi. This protein, with a cytosol-exposed epitope, offers a new tool for studying the cis-Golgi region.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cis-Golgi region is crucial for protein modification and sorting but remains poorly characterized due to a lack of specific markers.
- Integral membrane proteins are vital components of cellular organelles, yet their specific roles in the Golgi remain incompletely understood.
Purpose of the Study:
- To identify and characterize novel integral membrane proteins of the Golgi apparatus.
- To develop a tool for investigating the structure and function of the cis-Golgi.
Main Methods:
- Generation of a monoclonal antibody (mAb HFD9) against a Golgi-enriched protein.
- Immunofluorescence microscopy and immunogold labeling to determine protein localization.
- Biochemical extraction methods to assess membrane integration.
Main Results:
- A novel 28 kDa integral membrane protein (p28) was identified, localized to the cis-Golgi.
- The epitope recognized by mAb HFD9 is exposed to the cytosol.
- p28 is highly conserved and enriched in the Golgi membrane.
Conclusions:
- Monoclonal antibody HFD9 defines a new, cytoplasmically-oriented integral membrane protein of the cis-Golgi.
- p28 is the first identified Golgi integral membrane protein with a cytosolic epitope, providing a valuable marker for the cis-Golgi.
- This discovery facilitates further research into the under-characterized cis-Golgi network.