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Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
Published on: January 15, 2016
Monoclonal antibodies reactive with the BAF155 (SMARCC1) and BAF170 (SMARCC2) components of human SWI/SNF-related
Xiaomei Wang1, Da-Wei Liao, Michael Van Scoy
1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Abstract:
Mammalian SWI/SNF-related chromatin remodeling complexes are required for transcription controls that underlie differentiation, development, and tumor suppression. The complexes each consist of an ATPase of the SWI2/SNF2 family and approximately seven stably associated non-catalytic subunits. In spite of the importance of these complexes to biological processes, monoclonal antibodies to the various subunits have not been readily available. Mammalian complexes can vary in subunit composition, but the BAF155 (SMARCC1) subunit and a closely related protein, BAF170 (SMARCC2), appear to be ubiquitous components. Here we report the development of antibodies raised against a BAF155-derived peptide. The antibodies were raised against a single peptide of 18 amino acids. However, hybridomas expressing antibodies of two different specificities were isolated. One, designated DXD7, is specific for BAF155. The other, designated DXD12, is reactive with both BAF155 and BAF170. The antibodies are reactive against both native and denatured proteins, and are suitable for immunoprecipitation and Western blots. The DXD7 antibody is suitable additionally for immunofluorescence assays.
Insights
Researchers developed new antibodies targeting BAF155 and BAF170 proteins, crucial for chromatin remodeling complexes involved in cell processes. These tools aid in studying gene regulation and development.
Area of Science:
- Molecular Biology
- Cell Biology
- Epigenetics
Background:
- Mammalian SWI/SNF complexes regulate critical cellular functions like differentiation, development, and tumor suppression.
- These complexes comprise a catalytic ATPase subunit and approximately seven non-catalytic subunits, but specific antibodies for subunits have been scarce.
- BAF155 (SMARCC1) and BAF170 (SMARCC2) are ubiquitous subunits in mammalian SWI/SNF complexes.
Purpose of the Study:
- To develop novel monoclonal antibodies for specific subunits of mammalian SWI/SNF-related chromatin remodeling complexes.
- To generate tools for investigating the roles of BAF155 and BAF170 in biological processes.
Main Methods:
- Antibodies were generated by immunizing mice with a synthetic peptide derived from BAF155.
- Hybridoma technology was employed to isolate antibody-producing cell lines.
- Antibody specificity was determined using various biochemical and immunological assays, including Western blotting and immunoprecipitation.
Main Results:
- Two distinct hybridomas, DXD7 and DXD12, were successfully developed.
- DXD7 antibody specifically recognizes BAF155.
- DXD12 antibody reacts with both BAF155 and BAF170.
- Both antibodies are effective against native and denatured proteins and suitable for immunoprecipitation and Western blotting.
- The DXD7 antibody is also validated for immunofluorescence assays.
Conclusions:
- Novel monoclonal antibodies, DXD7 and DXD12, targeting BAF155 and BAF170, have been successfully developed.
- These antibodies provide valuable tools for studying the composition and function of mammalian SWI/SNF complexes.
- The availability of these reagents will facilitate research into chromatin remodeling, gene transcription, and related cellular processes.
