|96128T||1 Kit (6 x 20 microliters)||
|CD9 (E8L5J) Rabbit mAb 98327||20 µl||
||M R||22-27||Rabbit IgG|
|CD81 (D5O2Q) Rabbit mAb 10037||20 µl||
|TSG101 (E6V1X) Rabbit mAb 72312||20 µl||
||H M R||50||Rabbit IgG|
|Alix (E6P9B) Rabbit mAb 92880||20 µl||
||H M R||90-100||Rabbit IgG|
|Flotillin-1 (D2V7J) XP® Rabbit mAb 18634||20 µl||
||H M R||49||Rabbit IgG|
|HSP70 Antibody 4872||20 µl||
||H M R Mk B||72, 73||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu119 of mouse CD9, Pro184 of mouse CD81, Pro346 of human Alix, Ile368 of human flotillin-1, and residues near the amino terminus of human TSG101. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to human HSP70. Antibodies are purified by protein A and peptide affinity chromatography.
Exosomes are small (30-150 nm) membrane-bound vesicles that are secreted by various cell types under normal and pathological conditions (1,2). They originate from intracellular multivesicular endosomes upon fusion with the plasma membrane. Exosomes have emerged as an important mechanism of intercellular communication facilitating the transfer of membrane and cytosolic proteins, lipids, and RNA.
A variety of methods have been described to isolate exosomes and understand their composition (3-7). Heterogeneity in exosome composition can be attributed to the cells of origin as well as the isolation methods. However, there are protein markers that appear with high frequency. Tetraspanins are a family of cell surface glycoproteins with four transmembrane domains often found in exosomes (8). Tetraspanins CD9, CD81, and CD63 appear in exosomes and have been the target of immune-affinity approaches of exosome isolation. Flotillin-1 is a lipid raft-associated integral membrane protein that is incorporated into exosomes (9). Exosomes also contain proteins involved in endosomal membrane trafficking, collectively known as the ESCRT (endosomal sorting complex required for transport) pathway. Alix regulates cellular processes, such as endocytic membrane trafficking and cell adhesion through interactions with ESCRT proteins including endophilins, and CIN85 (Cbl-interacting protein of 85 kDa), and plays a role in exosome biogenesis (10-12). Syntenin-1 (MDA9, SDCBP) is a member of the PDZ family of proteins that functions as a scaffold adaptor protein regulating numerous signal transduction pathways (13). Syntenin-1 interacts with Alix to regulate exosome biogenesis (12). Tumor susceptibility gene 101 (TSG101) is a fundamental component of the ESCRT complex I involved in regulating the trafficking of proteins throughout the endosomal compartment (14). TSG101 is involved in regulating diverse biological processes, such as cell proliferation, viral budding and release, and exosome biosynthesis (15,16). The heat shock protein HSP70 is a molecular chaperone involved in protein folding that can be induced upon environmental stress (17). HSP70 may also be secreted through exosomes (18).
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