#74220 Exosomal Marker Antibody Sampler Kit
|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-P||H||89, 92||Rabbit|
|CD9 (D8O1A) Rabbit mAb #13174||20 µl||WB||H||22, 24, 35||Rabbit IgG|
|GM130 (D6B1) XP® Rabbit mAb #12480||20 µl||WB, IP, IF-IC||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-P, IF-IC||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|
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey
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.
Western blot analysis of extracts from Ramos, A549 and Raji cells, untreated (-) or TNF-α-treated (+), using CD54/ICAM-1 Antibody.
Western blot analysis of extracts from HeLa, NIH/3T3 and C6 cells, using HSP70 (D69) Antibody.
Western blot analysis of cell extracts from various cell types using Alix (3A9) Mouse mAb.
Western blot analysis of extracts from various cell lines using Annexin V Antibody.
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).
Western blot analysis of extracts from various cell lines using GM130 (D6B1) XP® Rabbit mAb.
Western blot analysis of extracts from various cell lines using CD9 (D8O1A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from various cell lines using Flotillin-1 (D2V7J) XP® Rabbit mAb.
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).
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).
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.
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.
Immunohistochemical analysis of paraffin-embedded Raji cells using CD54/ICAM-1 Antibody.
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.
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).
Confocal immunofluorescent analysis of BT-20 cells using Flotillin-1 (D2V7J) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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).
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The Annexin V-FITC conjugate is a product manufactured for Cell Signaling Technology, Inc. by TAU Technologies BV.
Tween is a registered trademark of ICI Americas, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
SignalStain is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.