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74220
Exosomal Marker Antibody Sampler Kit

Exosomal Marker Antibody Sampler Kit #74220

Western Blotting Image 1

Western blot analysis of cell extracts from various cell types using Alix (3A9) Mouse mAb.

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Western Blotting Image 2

Western blot analysis of extracts from various cell lines using Annexin V Antibody.

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Western Blotting Image 3

Western blot analysis of extracts from Ramos, A549 and Raji cells, untreated (-) or TNF-α-treated (+), using CD54/ICAM-1 Antibody.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines using CD9 (D8O1A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using GM130 (D6B1) XP® Rabbit mAb.

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Western Blotting Image 6

Western blot analysis of extracts from MCF7 (EpCAM positive), HT-29 (EpCAM positive), and HeLa (EpCAM negative) cells using EpCAM (D1B3) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 7

Western blot analysis of extracts from HeLa, NIH/3T3 and C6 cells, using HSP70 (D69) Antibody.

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using Flotillin-1 (D2V7J) XP® Rabbit mAb.

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Western Blotting Image 9

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.

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IHC-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded human lymph node using CD54/ICAM-1 Antibody in the presence of control peptide (left) or antigen specific peptide (right).

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Western Blotting Image 11

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human CD9 protein (hCD9-Myc/DDK; +), using CD9 (D8O1A) Rabbit mAb.

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IF-IC Image 12

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Brefeldin A #9972 (200 μM, 30 min; right), using GM130 (D6B1) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IP Image 13

Immunoprecipitation of flotillin-1 protein from BT-20 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Flotillin-1 (D2V7J) XP® Rabbit mAb. Western blot analysis was performed using Flotillin-1 (D2V7J) XP® Rabbit mAb.

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IHC-P (paraffin) Image 14

Immunohistochemical analysis of paraffin-embedded Raji cells using CD54/ICAM-1 Antibody.

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IF-IC Image 15

Confocal immunofluorescent analysis of HCT 116 (positive, left) and A498 (negative, right) cells, using CD9 (D8O1A) Rabbit mAb (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).

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 Image 16

Immunoprecipitation of GM130 protein from ZR-75-1 cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or GM130 (D6B1) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using GM130 (D6B1) XP® Rabbit mAb.

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using Flotillin-1 (D2V7J) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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IF-IC Image 18

Confocal immunofluorescent analysis of BT-20 cells using Flotillin-1 (D2V7J) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Alix (3A9) Mouse mAb 2171 20 µl
  • WB
  • IP
H M R Mk 95 Mouse IgG1
Annexin V Antibody 8555 20 µl
  • WB
H M R Mk 30 Rabbit 
CD54/ICAM-1 Antibody 4915 20 µl
  • WB
  • IHC
H 89, 92 Rabbit 
CD9 (D8O1A) Rabbit mAb 13174 20 µl
  • WB
  • IF
H 22, 24, 35 Rabbit IgG
GM130 (D6B1) XP® Rabbit mAb 12480 20 µl
  • WB
  • IP
  • IF
H Mk 130 Rabbit IgG
EpCAM (D1B3) Rabbit mAb 2626 20 µl
  • WB
  • IP
H 40 Rabbit IgG
HSP70 (D69) Antibody 4876 20 µl
  • WB
H M R Mk 70 Rabbit 
Flotillin-1 (D2V7J) XP® Rabbit mAb 18634 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R 49 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

The Exosomal Marker Antibody Sampler Kit provides an economical means to evaluate the presence of exosomal markers. The kit includes enough primary antibody to perform two western blot experiments for each target.

All antibodies provided in the kit detect endogenous levels of the respecitve target protein. Additionally, the Annexin V Antibody is not predicted to cross-react with other annexin family members and the CD54/ICAM-1 Antibody does not cross-react with other IgSF adhesion molecules. The GM130 antibody may cross-react with a protein of unknown origin at 30 kDa.

Monoclonal antibodies are produced by immunizing animals with full-length recombinant human Alix protein, a synthetic peptide corresponding to residues surrounding Val178 of human CD9 protein, residues surrounding Thr195 of human GM130 protein, residues near the amino terminus of human EpCAM protein, and residues surrounding Ile368 of human flotillin-1 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human annexin V protein, residues of human CD54 (ICAM-1) protein, and residues surrounding Asp69 of human HSP70. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Exosomes are small membrane-bound vesicles that in recent years have emerged as important molecules for inter-cellular communication. Exosomes are produced during both normal and pathophysiological conditions, and cancer cells have been shown to secrete exosomes in greater amounts than normal cells (reviewed in 1). The exosomal markers contained in this kit are Alix, Annexin V, ICAM-1, CD9, GM130, EpCAM, flotillin, and HSP70.

Alix, a cytosolic scaffold protein, regulates many cellular processes including endocytic membrane trafficking, cell adhesion through interactions with ESCRT (endosomal sorting complex required for transport) proteins, endophilins, and CIN85 (Cbl-Interacting protein of 85 kDa) (2, 3).

Annexin V is a ~30 kDa protein that binds to phospho-lipids in a calcium-dependent manner (4). All annexins contain a putative PKC binding site, but only annexin V has been identified as an inhibitor of this pathway (5).

Intracellular cell adhesion molecule-1 (CD54 or ICAM-1) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily (IgSF) of adhesion molecules. CD54 is expressed at low levels in diverse cell types, and is induced by cytokines (TNF-alpha, interleukin-1) and bacterial lipopolysaccharides (6). Apical localization on endothelial cells (or basolateral localization on epithelial cells) is a prerequisite for leukocyte trafficking through the endothelial (or epithelial) barrier (6).

The CD9 antigen belongs to the tetraspanin family of cell surface glycoproteins. Tetraspanins interact with a variety of cell surface proteins and intracellular signaling molecules in specialized tetraspanin-enriched microdomains (TEMs), where they mediate a range of processes including adhesion, motility, membrane organization, and signal transduction (7). Additional research identified CD9 as an abudant component of exosomes, and may play a role in the fusion of these secreted membrane vesicles with recipient cells (8).

GM130 is required for membrane fusion events that mediate ribbon formation during Golgi assembly (9). The Golgi apparatus functions in the modification, organization, and transport of proteins and membrane targeted to other parts of the cell, such as the plasma membrane, lysosomes, and endosomes. This regulated transport is important for appropriate protein localization, secretion, and signal transduction (reviewed in 10).

Epithelial cell adhesion and activation molecule (EpCAM/CD326) is a transmembrane glycoprotein that mediates calcium-independent, hemophilic adhesions on the basolateral surface of most epithelial cells (11). One of the first tumor-associated antigens discovered, EpCAM has long been a marker of epithelial and tumor tissue. Research studies have shown that EpCAM is highly expressed in cancer cells and can be used as a biomarker for the detectionof tumor-derived exposomes (reviewed in 1, 12, 13).

Flotillins belong to a famiy of lipid raft-associated integral membrane proteins that are ubiquitously expressed and located to lipid rafts on the cell plasma membrane where they support signal transduction and regulate lipid raft motility and localization (14-17). In addition to its colocalization with lipid rafts on the plasma membrane, flotillin-1 also has been found at compartments of the endocytic and autophagosomal pathways, such as recycling/ late endosomes, the Golgi complex, as well as the nucleus (18, 19).

HSP70 is a molecular chaperone expressed constituitively under normal conditions to maintain protein homeostatis and is induced upon environmental stress (20). HSP70 is able to interact with unfolded proteins to prevent irreversible aggregation and catalyze the refolding of their substrates in an ATP and co-chaperone dependent manner (21). An immune response is elicited upon excretion of heat shock proteins from tumor exosomes (reviewed in 1).

  1. Raposo, G. and Stoorvogel, W. (2013) J Cell Biol 200, 373-83.
  2. Huber, R. et al. (1990) EMBO J 9, 3867-74.
  3. Hopkins, A.M. et al. (2004) Adv Drug Deliv Rev 56, 763-78.
  4. Hemler, M.E. (2005) Nat Rev Mol Cell Biol 6, 801-11.
  5. Barr, F.A. and Short, B. (2003) Curr Opin Cell Biol 15, 405-13.
  6. Went, P.T. et al. (2004) Hum Pathol 35, 122-8.
  7. Nollen, E.A. and Morimoto, R.I. (2002) J. Cell Sci. 115, 2809-2816.
  8. Cardó-Vila, M. et al. (2003) Mol Cell 11, 1151-62.
  9. Puthenveedu, M.A. et al. (2006) Nat Cell Biol 8, 238-48.
  10. Baeuerle, P.A. and Gires, O. (2007) Br J Cancer 96, 417-23.
  11. Langhorst, M.F. et al. (2005) Cell Mol Life Sci 62, 2228-40.
  12. Young, J.C. et al. (2003) Trends Biochem. Sci. 28, 541-547.
  13. Armstrong, A. and Eck, S.L. (2003) Cancer Biol Ther 2, 320-6.
  14. Stuermer, C.A. and Plattner, H. (2005) Biochem Soc Symp , 109-18.
  15. Fernow, I. et al. (2007) Eur J Cell Biol 86, 345-52.
  16. Katoh, K. et al. (2003) J. Biol. Chem. 278, 39104-39113.
  17. Neumann-Giesen, C. et al. (2007) J Cell Sci 120, 395-406.
  18. Sadoul, R. (2006) Biol. Cell 98, 69-77.
  19. Liu, J. et al. (2005) J Biol Chem 280, 16125-34.
  20. Santamaría, A. et al. (2005) Mol Cell Biol 25, 1900-11.
  21. Théry, C. et al. (1999) J Cell Biol 147, 599-610.
Entrez-Gene Id
10015 , 308 , 928 , 4072 , 10211 , 2801 , 3303 , 3383
Swiss-Prot Acc.
Q8WUM4 , P08758 , P21926 , P16422 , O75955 , Q08379 , P0DMV8 , P05362
For Research Use Only. Not For Use In Diagnostic Procedures.

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XP is a registered trademark of Cell Signaling Technology, Inc.

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