Revision 1
Cell Signaling Technology

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Pan-Keratin (C11) Mouse mAb 4545 40 µl 46-58 kDa Mouse IgG1
GFAP (GA5) Mouse mAb 3670 40 µl 50 kDa Mouse IgG1
Vimentin (D21H3) XP® Rabbit mAb 5741 40 µl 57 kDa Rabbit IgG
Desmin (D93F5) XP® Rabbit mAb 5332 40 µl 53 kDa Rabbit IgG
Plectin-1 (D6A11) Rabbit mAb 12254 40 µl 400-500 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

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).

  1. Eng, L.F. et al. (2000) Neurochem Res 25, 1439-51.
  2. Goebel, H.H. et al. (1987) Acta Histochem Suppl 34, 81-93.
  3. Leader, M. et al. (1987) Histopathology 11, 63-72.
  4. Capetanaki, Y. et al. (2007) Exp Cell Res 313, 2063-76.
  5. Li, Z. et al. (1996) Dev Biol 175, 362-6.
  6. Paulin, D. and Li, Z. (2004) Exp Cell Res 301, 1-7.
  7. Paulin, D. et al. (2004) J Pathol 204, 418-27.
  8. Moll, R. et al. (1982) Cell 31, 11-24.
  9. Chang, L. and Goldman, R.D. (2004) Nat Rev Mol Cell Biol 5, 601-13.
  10. Ramaekers, F.C. and Bosman, F.T. (2004) J Pathol 204, 351-4.
  11. Lane, E.B. and McLean, W.H. (2004) J Pathol 204, 355-66.
  12. Zatloukal, K. et al. (2004) J Pathol 204, 367-76.
  13. Owens, D.W. and Lane, E.B. (2004) J Pathol 204, 377-85.
  14. Wiche, G. (1998) J Cell Sci 111 ( Pt 17), 2477-86.
  15. Osmanagic-Myers, S. and Wiche, G. (2004) J Biol Chem 279, 18701-10.
  16. Osmanagic-Myers, S. et al. (2006) J Cell Biol 174, 557-68.
  17. Ding, Y. et al. (2008) Exp Cell Res 314, 590-602.
  18. Gregor, M. et al. (2006) J Cell Sci 119, 1864-75.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #4751

    Intermediate Filaments Antibody Sampler Kit

    Intermediate Filaments Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse kidney labeled with Vimentin (D21H3) XP® Rabbit mAb (left, green) and co-labeled with F4/80 (BM8.1) Rat mAb (right, red), and ProLong® Gold Antifade Reagent with DAPI #8961 (right, blue).
    Intermediate Filaments Antibody Sampler Kit: Image 2 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse colon labeled with Vimentin (D21H3) XP® Rabbit mAb (left, green) and co-labeled with F4/80 (BM8.1) Rat mAb (right, red), and ProLong® Gold Antifade Reagent with DAPI #8961 (right, blue).
    Intermediate Filaments Antibody Sampler Kit: Image 3 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse cerebellum labeled with Vimentin (D21H3) XP® Rabbit mAb (left, green) and co-labeled with F4/80 (BM8.1) Rat mAb (right, red), and ProLong® Gold Antifade Reagent with DAPI #8961 (right, blue).
    Intermediate Filaments Antibody Sampler Kit: Image 4 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from HeLa cells using Vimentin (D21H3) XP ® Rabbit mAb #5741. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Intermediate Filaments Antibody Sampler Kit: Image 5 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse cerebellum labeled with Vimentin (D21H3) XP® Rabbit mAb (left, green) and co-labeled with F4/80 (BM8.1) Rat mAb (right, red), and ProLong® Gold Antifade Reagent with DAPI #8961 (right, blue).
    Intermediate Filaments Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from COS-7 and U-2 OS cells using Plectin-1 (D6A11) Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from mouse and rat brain, using GFAP (GA5) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from various cell lines, using Pan-Keratin (C11) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from C2C12 cells, rat heart and human heart using Desmin (D93F5) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using Vimentin (D21H3) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 11 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Intermediate Filaments Antibody Sampler Kit: Image 12 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
    Intermediate Filaments Antibody Sampler Kit: Image 13 Expand Image
    Immunoprecipitation of plectin-1 from U-2 OS cell extracts, using Plectin-1 (D6A11) Rabbit mAb (lane 2) or Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Plectin-1 (D6A11) Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human medulloblastoma, using GFAP (GA5) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma (bladder), using Pan-Keratin (C11) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 16 Expand Image
    Confocal immunofluorescent analysis of mouse skeletal muscle using Desmin (D93F5) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Intermediate Filaments Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or Vimentin knockout HeLa cells (lane 2) using Vimentin (D21H3) XP® Rabbit mAb #5741 (upper) or β-Actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the Vimentin knockout HeLa cells confirms specificity of the antibody for Vimentin.
    Intermediate Filaments Antibody Sampler Kit: Image 18 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Plectin-1 (D6A11) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Intermediate Filaments Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescence image of rat hippocampus labeled with GFAP (GA5) Mouse mAb (red), Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mAb (Alexa Fluor® 488 Conjugate) #4854 (green), and CREB (48H2) Rabbit mAb #9197 (blue).
    Intermediate Filaments Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Pan-Keratin (C11) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 21 Expand Image
    Confocal immunofluorescent analysis of choroid plexus in mouse brain using Desmin (D93F5) XP® Rabbit mAb (green). After blocking free secondary antibody binding sites with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, the tissue was then labeled using Ras (E4K9L) Rabbit mAb (Alexa Fluor® 647 Conjugate) #37182 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Intermediate Filaments Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Vimentin (D21H3) XP® Rabbit mAb performed on the Leica® BOND Rx.
    Intermediate Filaments Antibody Sampler Kit: Image 23 Expand Image
    Confocal immunofluorescent analysis of SNB19 (positive, left) and HeLa (negative, right) cells, using GFAP (GA5) Mouse mAb (green). Actin filaments were labeled with DyLight® 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Intermediate Filaments Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Pan-Keratin (C11) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 25 Expand Image
    Confocal immunofluorescent analysis of mouse liver at low (left) or high magnification (right) using Desmin (D93F5) XP® Rabbit mAb (green). After blocking free secondary antibody binding sites with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, the tissue was then labeled using Ras (E4K9L) Rabbit mAb (Alexa Fluor® 647 Conjugate) #37182 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Intermediate Filaments Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Vimentin (D21H3) XP® Rabbit mAb performed on the Leica® BOND Rx.
    Intermediate Filaments Antibody Sampler Kit: Image 27 Expand Image
    Flow cytometric analysis of HeLa cells (blue) and SNB-19 cells (green) using GFAP (GA5) Mouse mAb (solid lines) or a concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
    Intermediate Filaments Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Pan-Keratin (C11) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of mouse pancreas using Desmin (D93F5) XP® Rabbit mAb (green). After blocking free secondary antibody binding sites with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, the tissue was then labeled using Ras (E4K9L) Rabbit mAb (Alexa Fluor® 647 Conjugate) #37182 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Intermediate Filaments Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Vimentin (D21H3) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded H358 xenograft, using Pan-Keratin (C11) Mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using Vimentin (D21H3) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Pan-Keratin (C11) mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 34 Expand Image
    Confocal immunofluorescent analysis of C2C12 cells (left, positive) or Neuro-2a cells (right, negative), using Desmin (D93F5) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Intermediate Filaments Antibody Sampler Kit: Image 35 Expand Image
    Immunohistochemical analysis of paraffin-embedded rat colon using Vimentin (D21H3) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Pan-Keratin (C11) mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 37 Expand Image
    Confocal immunofluorescent analysis of SNB19 cells using Vimentin (D21H3) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Intermediate Filaments Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded rhesus kidney using Vimentin (D21H3) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 39 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Pan-Keratin (C11) mouse mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 40 Expand Image
    Flow cytometric analysis of MCF7 cells (blue, negative) and HeLa cells (green, positive) using Vimentin (D21H3) XP® Rabbit mAb(solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Intermediate Filaments Antibody Sampler Kit: Image 41 Expand Image
    Immunohistochemical analysis of paraffin-embedded Syrian hamster small intestine using Vimentin (D21H3) XP® Rabbit mAb.
    Intermediate Filaments Antibody Sampler Kit: Image 42 Expand Image
    Immunohistochemical analysis of paraffin-embedded human tonsil using Vimentin (D21H3) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Intermediate Filaments Antibody Sampler Kit: Image 43 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Pan-Keratin (C11) Mouse mAb (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
    Intermediate Filaments Antibody Sampler Kit: Image 44 Expand Image
    Flow cytometric analysis of A-431 cells using Pan-Keratin (C11) Mouse mAb (solid line) versus a concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed line). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.