The Intermediate Filaments Antibody Sampler Kit provides an economical means to evaluate the presence and status of intermediate filaments. The kit includes enough primary and secondary antibody to perform four Western blot experiments per antibody.
Pan-Keratin (C11) Mouse mAb detects endogenous levels of total keratin 4, 5, 6, 8, 10, 13 and 18 and does not cross-react with other keratins. Plectin-1 (D6A11) Rabbit mAb recognizes endogenous levels of total plectin-1 protein. GFAP (GA5) Mouse mAb detects endogenous levels of total GFAP protein. Desmin Antibody detects endogenous levels of total desmin protein. Vimentin (R28) Antibody detects endogenous levels of total vimentin protein.
Pan-Keratin (C11) monoclonal antibody is produced by immunizing animals with a cytoskeleton preparation from A431 cells. GFAP (GA5) monoclonal antibody is produced by immunizing animals with native GFAP purified from pig spinal cord. Desmin monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to carboxy terminal residues of human desmin protein, and Plectin-1 (D6A11) monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu2980 of human plectin-1 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg28 of human vimentin. Antibodies are purified by peptide affinity chromatography.
The cytoskeleton consists of three types of cytosolic fibers: microfilaments (actin filaments), intermediate filaments and microtubules. Major types of intermediate filaments are distinguished and expressed in particular cell types: cytokeratins (epithelial cells), glial fibrillary acidic protein, GFAP (glial cells), desmin (skeletal, visceral and certain vascular smooth muscle cells), vimentin (mesenchyme origin) and neurofilaments (neurons). GFAP and vimentin form intermediate filaments in astroglial cells and modulate their motility and shape (1). In particular, vimentin filaments are present at early developmental stages, while GFAP filaments are characteristic of differentiated and mature brain astrocytes. Thus, GFAP is commonly used as a marker for intracranial and intraspinal tumors arising from astrocytes (2). Vimentin is present in sarcomas, but not carcinomas, and its expression is examined in conjunction with that of other markers to distinguish between the two (3).
Desmin is a myogenic marker expressed in early development that forms a network of filaments that extends across the myofibril and surrounds Z discs. The desmin cytoskeleton provides a connection between myofibrils, organelles and the cytoskeleton (4). Desmin knockout mice develop cardiomyopathy as well as skeletal and smooth muscle defects (5). In humans, desmin related myopathies might be caused by mutations in the corresponding desmin gene or in proteins with which desmin interacts, including αB-crystallin and synemin. Disorganized desmin filaments and the accumulation of protein aggregates comprised predominantly of desmin characterize desmin-related myopathies (reviewed in 6,7).
Keratins assemble into filaments, forming heterodimers of an acidic keratin (or type I keratin, keratins 9 to 23) and a basic keratin (or type II keratin, keratins 1 to 8) (8,9). Keratin isoforms demonstrate tissue- and differentiation-specific profiles, which make them useful as biomarkers (8). Mutations in keratin genes are associated with skin disorders, liver and pancreatic diseases, and inflammatory intestinal diseases (10-13).
Plectin is a large, widely expressed protein that crosslinks the intermediate filament and actin cytoskeleton, mechanically stabilizing cells and tissues. Plectin also plays a role in the regulation of actin dynamics and acts as a scaffold for signaling molecules (14). It is important in the stabilization of hemidesmosomes, crosslinking them to the intermediate filament network. Plectin has been shown to be involved in several signaling cascades. It signals to PKC by binding to and sequestering RACK1, the receptor for activated C kinase 1 (15,16). Plectin is also involved in the regulation of cytokeratin architecture and cell stress response (16), signaling through the chemokine receptor CXCR4 (17), regulation of AMP-activated protein kinase (AMPK) activity and signaling in mouse myotubes (18).
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