Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H||WB, IHC, IF||M||Purified||Monoclonal Antibody|
|Description||Anti-Nuclear Spliceosomes Antibody, clone 780-3|
|Presentation||Hybridoma supernatant, concentrated by ammonium sulfate. Buffer: PBS with 0.1% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain at -20°C in undiluted aliquots for up to 12 months. Avoid repeated freeze/thaw cycles.|
|Material Size||100 µL|
|Reference overview||Pub Med ID|
|The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes. |
Kumar, P; Kumar, P; Lindberg, L; Thirkill, TL; Ji, JW; Martsching, L; Douglas, GC
PloS one 7 e42712 2012
MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C) can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N) does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters) and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB). While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC1-C have dissimilar intranuclear distribution patterns.
|Quantitative analysis of a nuclear antigen in interphase and mitotic cells. |
Clevenger, C V, et al.
Cytometry, 8: 280-6 (1987) 1987
The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.
|Modulation of the nuclear antigen p105 as a function of cell-cycle progression. |
Clevenger, C V, et al.
J. Cell. Physiol., 130: 336-43 (1987) 1987
The characterization of the proliferation-associated nuclear antigen designated p105 in quiescent and proliferating lymphocytes is described. Through the use of novel flow cytometric and cell-sorting strategies the intracellular content of p105 was assessed in situ on a per cell basis. These analyses demonstrated the presence of multiple cellular subpopulations within the cell cycle differing significantly in p105 content. The data revealed that the flow cytometric quantitation of p105 levels may effectively discriminate cycling from noncycling cells. Immunogold electron microscopy revealed that the modulation of this interchromatin-associated antigen was correlated with a significant degree of nuclear restructuring. In conjunction with cell sorting, immunogold electron microscopy and immunoblot controls demonstrated that the cell-cycle-related modulation in p105 cannot be accounted for by increased cellular mass or antigen sequestration. The significance of these controls and of the potential role of p105 in cellular proliferation is discussed.
|Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry. |
Bauer, K D, et al.
Cancer Res., 46: 2428-34 (1986) 1986
The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G1-G0 cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.
|A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry. |
Clevenger, C V, et al.
Cytometry, 6: 208-14 (1985) 1985
A preparative technique for the two-parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4 degrees C first with 0.5% paraformaldehyde and second with 0.1% Triton X-100 in phosphate-buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high-quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen-specific monoclonal antibodies in conjunction with this technique, the cell-cycle phase-dependent expression of such antigens is examined. From these data, the utility of two-parameter flow cytometry in the identification and quantification of cell-cycle-dependent modulation of nuclear antigens is discussed.
|Identification of a nuclear protein component of interchromatin granules using a monoclonal antibody and immunogold electron microscopy. |
Clevenger, C V and Epstein, A L
Exp. Cell Res., 151: 194-207 (1984) 1984
A monoclonal antibody, designated 780-3, has been generated which preferentially recognizes an antigenic component of interchromatin granules in human cells. By indirect immunofluorescence procedures, monoclonal antibody 780-3 produces a cell cycle-specific speckled nuclear staining pattern in adult human fibroblasts which is dramatically altered during metaphase. In contrast, transformed cells appear to express this antigen throughout the cell cycle in increased quantities. Immunogold electron microscopy revealed that the nuclear antigen is intimately associated with interchromatin granules in human cells. Analysis by immunoblot procedures showed that monoclonal antibody 780-3 recognizes two polypeptides of 105 and 41 kD. From these data, a possible nucleolar derivation of interchromatin granules is discussed. These studies demonstrate for the first time that monoclonal antibodies may be used in combination with immunogold electron microscopy to identify the ultrastructural location of nuclear antigens.
|Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures. |
Clevenger, C V and Epstein, A L
J. Histochem. Cytochem., 32: 757-65 (1984) 1984
A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.
|MOUSE ANTI-HUMAN NUCLEAR SPLICEOSOMES MONOCLONAL ANTIBODY - Data Sheet|