Cd63 mx 49

Manufactured by Santa Cruz Biotechnology

Sourced in United States

CD63 (MX-49.129.5) is a lab equipment product used for the detection and analysis of the CD63 protein. It functions as a marker for the identification and study of exosomes and other extracellular vesicles.

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Lab products found in correlation

Paxillin, by BD (1 mentions) Phospho akt s473, by Cell Signaling Technology (1 mentions) α tubulin, by Merck Group (1 mentions) Crkl c20, by Santa Cruz Biotechnology (1 mentions) Phospho erk1 2, by Cell Signaling Technology (1 mentions) P130cas, by BD (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) 12mm 1.5 glass coverslips, by Thermo Fisher Scientific (1 mentions) Elyra ps 1 microscope, by Zeiss (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions) Fluoromount g, by Southern Biotech (1 mentions) Ab126274, by Abcam (1 mentions) Ab97051, by Abcam (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Pvdf membrane, by GE Healthcare (1 mentions) Mabcam 9484, by Abcam (1 mentions) Af617, by R&D Systems (1 mentions) Cd81 5a6, by Santa Cruz Biotechnology (1 mentions) Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions)

Close spelling variants of this product

MX-49.129.5 (6 citations) Anti-CD63 (MX-49.129.5, (4 citations) CD63 (clone MX-49.129.5) (3 citations)

5 protocols using cd63 mx 49

Immunoblot Analysis of Signaling Proteins

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Immunoblot analyses were carried out as described previously.32, 39 The detailed protocols are described in Appendix S1. Antibodies against the following proteins were purchased. ErbB3 (C‐17), DOCK180 (H4), C3G (C19), Gab1, CRKL (C20), and CD63 (MX‐49.129.5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and paxillin, p130^Cas, and CRK were from BD Transduction Laboratories (Lexington, KY, USA). Epidermal growth factor receptor (EGFR), phospho‐EGFR (Y1148), c‐Met, phospho‐c‐Met (Y1234/1235), ErbB2 (29D8), focal adhesion kinase (FAK), phospho‐FAK (Y397), phospho‐Gab1 (Y307), Src, phospho‐Src (Y416), ERK1/2, phospho‐ERK1/2, Akt, and phospho‐Akt (S473) were from Cell Signaling Technology (Beverly, MA, USA). Actin was from Chemicon International (Temecula, CA, USA) and α‐tubulin was from Sigma‐Aldrich.

Yoshida K., Tsuda M., Matsumoto R., Semba S., Wang L., Sugino H., Tanino M., Kondo T., Tanabe K, & Tanaka S. (2019). Exosomes containing ErbB2/CRK induce vascular growth in premetastatic niches and promote metastasis of bladder cancer. Cancer Science, 110(7), 2119-2132.

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Imaging Platelet Activation and MRP4 Localization

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For imaging experiments, 12mm #1.5 glass coverslips (Fisher Scientific) were coated with poly-L-lysine (Sigma). Inhibitors
(MRS2395, 10 μM; ticagrelor, 20 ng/mL; PSB 0739, 10 μM; AR-C 66096, 10 μM) were added to platelets in
solution (4×10⁷/mL) for 15 min prior to activation with TRAP-6 (10 μM) for 30 sec at 37ºC,
followed by fixation with 4% paraformaldehyde and seeding onto proteins at room temperature for 1 hr. Adherent platelets were
permeabilized with a blocking solution (1% BSA + 0.01% SDS in PBS). Platelets were then stained with CD63 (MX49.129.5; Santa Cruz
Biotechnology) and MRP4/ABCC4 (D1Z3W; Cell Signaling Technology) overnight at 4ºC at a 1:100 dilution in the blocking
solution. Alexa Fluor secondary antibodies (1:500; Life Technology) were added in the blocking solution for 2 hrs. Coverslips were
mounted with Fluoromount G (SouthernBiotech) onto glass slides. Platelets were imaged using SR-SIM with a Zeiss 100× oil
immersion 1.46 NA alpha plan-apochromat lens on a Zeiss Elyra PS.1 microscope.

Mitrugno A., Rigg R.A., Laschober N.B., Ngo A.T., Pang J., Williams C.D., Aslan J.E, & McCarty O.J. (2017). Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395. Platelets, 29(4), 383-394.

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Exosome and Cell Lysate Protein Analysis

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Equal concentration of exosomes and cell lysates were loaded in mini-protean TGX precast gel (Bio-Rad) after addition of sample buffer and a heating step. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare). Blocking of the membranes was performed utilizing 3% ECL prime blocking agent (Amersham, GE Healthcare) for 1 h at room temperature. The membranes were exposed to an appropriate amount of primary antibodies such as CD40L (AF617, R&D Systems), 4-1BBL (ab126274, Abcam), GAPDH (mAbcam 9484, Abcam), CD63 (MX-49.129.5, Santa Cruz), or CD81 (5A6, Santa Cruz) and incubated at 4°C overnight while rotating. After a washing step, peroxidase-conjugated secondary antibodies such as mouse-immunoglobulin Gκ (IgGκ; BP-HRP, Santa Cruz), horseradish peroxidase (HRP)-conjugated anti-goat IgG (HAF109, R&S Systems), or HRP-conjugated anti-rabbit IgG (ab97051, Abcam) were added to the membranes, which were then washed with PBS/0.05% Tween 20 before development by enhanced chemiluminescence (ECL) prime western blotting detection reagents (Amersham, GE Healthcare). The protein bands were visualized by a ChemiDoc touch imaging system (Bio-Rad).

Labani-Motlagh A., Naseri S., Wenthe J., Eriksson E, & Loskog A. (2021). Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes. Molecular Therapy Oncolytics, 20, 508-518.

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Extracellular Vesicle Protein Profiling

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The filtrate sample was concentrated by ultrafiltration. Each sample was transferred to Amicon ultra 15 centrifugal filter units (100 kDa, Merck), and centrifugation was performed at 2000 rpm for 2 min at 4 °C. After measuring the number of vesicles in the samples via NTA, we prepared 2 × 10⁷ particles per sample for SDS-PAGE. The samples were boiled at 100 °C for 5 min with the 5× sample buffer and loaded them to 10% acrylamide gels and separated the proteins by SDS-PAGE. Then, the separated proteins in the gel were transferred onto 0.45 μm PVDF (polyvinylidene fluoride) membrane and subjected to immunoblotting with CD63 (MX-49.129.5, Santa Cruz), CD9 (EPR2949, Abcam), and syntenin (EPR8102, Abcam) antibody.

Kim D., Lee J., Kim B., Shin Y., Park J., Kim U., Lee M., Kim S.B, & Kim S. (2022). Ultra-thin membrane filter with a uniformly arrayed nanopore structure for nanoscale separation of extracellular vesicles without cake formation. Nanoscale Advances, 5(3), 640-649.

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Optimized Western Blot of Extracellular Vesicles

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Western blots were performed as described (69 ) with some modifications. Briefly, EVs were lysed in RIPA buffer with 1 mM PMSF; after 30 seconds’ brief sonication, samples were heated at 95°C for 5 minutes in sample loading buffer: reducing (0.1 M Tris-HCl at pH 6.8, 5% glycerol, 1.5% SDS, 1.3% DTT, and 0.04% bromophenol blue) or nonreducing sample buffer (without DTT). EVs were resolved by SDS-polyacrylamide gel electrophoresis, then transferred to nitrocellulose membranes and blotted with specific primary and secondary antibodies. Antibodies used for immunoblotting were purchased from Santa Cruz Biotechnology: CD63 (MX-49.129.5, sc-5275, nonreducing conditions), Calnexin (AF18, SC-23954), CD9 (ALB 6, sc-59140), HSP70 (W27, sc-24), Alix (1A12, sc-53540), TSG 101 (51, sc-136111), Albumin (F-10, sc-271605), CD81 (B-11, sc-166029), GAPDH (0411, sc-47724). All primary antibodies were diluted 1:1000, except GAPDH was diluted 1:30,000 and secondary antibody (m-IgGκ BP-HRP, sc-516102, diluted 1:2000). Protein bands were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) on x-ray film.

Elgamal S., Cocucci E., Sass E.J., Mo X.M., Blissett A.R., Calomeni E.P., Rogers K.A., Woyach J.A., Bhat S.A., Muthusamy N., Johnson A.J., Larkin K.T, & Byrd J.C. (2021). Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant. JCI Insight, 6(15), e137937.

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