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4751
Intermediate Filaments Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Intermediate Filaments Antibody Sampler Kit #4751

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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).
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).
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).
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.
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).
Western blot analysis of extracts from COS-7 and U-2 OS cells using Plectin-1 (D6A11) Rabbit mAb.
Western blot analysis of extracts from mouse and rat brain, using GFAP (GA5) Mouse mAb.
Western blot analysis of extracts from various cell lines, using Pan-Keratin (C11) Mouse mAb.
Western blot analysis of extracts from C2C12 cells, rat heart and human heart using Desmin (D93F5) XP® Rabbit mAb.
Western blot analysis of extracts from various cell lines using Vimentin (D21H3) XP® Rabbit mAb.
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.
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.
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.
Immunohistochemical analysis of paraffin-embedded human medulloblastoma, using GFAP (GA5) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma (bladder), using Pan-Keratin (C11) Mouse mAb.
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).
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.
Confocal immunofluorescent analysis of HeLa cells using Plectin-1 (D6A11) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Pan-Keratin (C11) Mouse mAb.
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).
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Vimentin (D21H3) XP® Rabbit mAb performed on the Leica® BOND Rx.
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).
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Pan-Keratin (C11) Mouse mAb.
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).
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Vimentin (D21H3) XP® Rabbit mAb performed on the Leica® BOND Rx.
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.
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Pan-Keratin (C11) Mouse mAb.
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).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Vimentin (D21H3) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded H358 xenograft, using Pan-Keratin (C11) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded mouse colon using Vimentin (D21H3) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Pan-Keratin (C11) mouse mAb.
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).
Immunohistochemical analysis of paraffin-embedded rat colon using Vimentin (D21H3) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Pan-Keratin (C11) mouse mAb.
Confocal immunofluorescent analysis of SNB19 cells using Vimentin (D21H3) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded rhesus kidney using Vimentin (D21H3) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Pan-Keratin (C11) mouse mAb.
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.
Immunohistochemical analysis of paraffin-embedded Syrian hamster small intestine using Vimentin (D21H3) XP® Rabbit mAb.
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).
Confocal immunofluorescent analysis of HeLa cells using Pan-Keratin (C11) Mouse mAb (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
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.
Inquiry Info.# 4751

Product 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.

Specificity / Sensitivity

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.

Source / Purification

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.

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.

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