Anti rab5b

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Anti-Rab5B is a polyclonal antibody that recognizes the Rab5B protein. Rab5B is a member of the Ras superfamily of small GTPases and is involved in the regulation of early endosome formation and function. The Anti-Rab5B antibody can be used for the detection of Rab5B in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.

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3 protocols using anti rab5b

Protein Extraction and Western Blot Analysis

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Whole-cell lysates and total exosomal proteins were prepared using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Fifteen micrograms total proteins were electrophoretically separated on NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific). Western blot analyses were performed with primary antibodies: anti-αSMA, anti-β-actin peroxidase-linked, anti-Vinculin, anti-αTubulin (1 : 500; 1 : 10 000 and 1 : 1000, respectively; Sigma-Aldrich, St. Louis, MO, USA), anti-CDH1, anti-Rab5B (1 : 1000; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Flot1 (1 : 1000; Cell Signalling, Boston, MA, USA), anti-Lamp2, anti-CD63, anti-COL1A1 (1 : 1000, BD Biosciences, Franklin Lakes, NJ, USA) and the corresponding secondary antibodies anti-mouse and anti-rabbit peroxidase-linked (1 : 10 000; GE Healthcare Life Sciences). The signals were visualized by ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences).

Baroni S., Romero-Cordoba S., Plantamura I., Dugo M., D'Ippolito E., Cataldo A., Cosentino G., Angeloni V., Rossini A., Daidone M.G, & Iorio M.V. (2016). Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts. Cell Death & Disease, 7(7), e2312-.

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Western Blot Analysis of Extracellular Vesicles

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Density gradient fractions were loaded on a 4–20% gradient Tris-Glycine precast polyacrylamide gel (Bio-Rad, Hercules, CA), separated by standard SDS-PAGE procedures and transferred onto PVDF membranes. (Immobilon, EMD Millipore, Billerica, MA). Primary antibodies used in this study were: anti-CD63 (1:1,000, #ab217345, Abcam, Cambridge, UK), anti-Alix (1:1,000, #ABC40, EMD Millipore), anti-TSG101 (1:1,000, #PA5-31260, ThermoFisher Scientific), anti-Rab35 (1:1,000, #9690, Cell Signaling Technology, Danvers, MA), anti-Rab5B (1:5,000, #sc-598, Santa Cruz Biotechnology, Dallas, TX), anti-Rab7 (1:5,000, #R8779, Sigma-Aldrich) and anti-EEA1 (1:1,000, #07-1820, EMD Millipore). The secondary antibodies used were HRP-conjugated anti-rabbit and anti-mouse antibodies from Jackson ImmunoResearch (West Grove, PA, US). The membranes were incubated in chemiluminescent fluid (Pierce, Rockford, IL, US) for 5 min, and chemiluminescence was visualized on Reflection Autoradiography films. Ponceau staining (Sigma-Aldrich) was used as a loading control. Protein bands were quantified through the open source software ImageJ (National Institute of Health (NIH), Bethesda, MD, US). Data are shown as the densitometric ratio between Ts2 and 2N controls, after normalization for protein concentration of each fraction.

D’Acunzo P., Hargash T., Pawlik M., Goulbourne C.N., Pérez-González R, & Levy E. (2019). Enhanced Generation of Intraluminal Vesicles in Neuronal Late Endosomes in the Brain of a Down Syndrome Mouse Model with Endosomal Dysfunction. Developmental neurobiology, 79(7), 656-663.

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Melanoma Cell Lines and Exosome Characterization

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All malignant melanoma cell lines were purchased from the American Type Tissue Collection (ATCC, Manassas, VA). MS1 murine endothelial cells [56 (link)] were a gift from Professor Kristian Pietras, (Lund University, Sweden). Recombinant proteins (rWNT5A (0.2 μg/ml) or rWNT3A (0.05 μg/ml) were from R&D systems, Minneapolis, MN. All chemicals and inhibitors were purchased from Sigma Aldrich (St Louis, MO.) unless otherwise noted and used at concentrations: 10 μM Bapta-AM or DMSO control, 5 μM PKA inhibitor H89, 10 ng/ml Tetanus Toxin or 10 μg/ml Brefeldin A. The antibodies used were Goat anti-WNT5A (R&D systems, Minneapolis, MN), anti-CD63, anti-Rab5B and anti-GAPDH (Santa Cruz biotechnologies), mouse anti-β-actin (MP biomedicals). For BD Cytometric Bead Array (CBA) the Human Inflammation Kit was used (BD Biosciences). For hematoxylin and eosin cells were fixed in 4% PFA and embedded in paraffin and then stained. F-actin cytoskeleton was stained using Alexafluor546-coupled Phalliodin. For ELISAs Quantikine Human IL-6 ELISA kit, Quantikine Human MMP2 ELISA kit and Quantikine Human VEGF ELISA kit from R&D systems (Minneapolis, MN) was used. Exosome Elisa (ExoELISA) CD63 ExoELISA^TM from System Biosciences (Uden, The Netherlands) was used.

Ekström E.J., Bergenfelz C., von Bülow V., Serifler F., Carlemalm E., Jönsson G., Andersson T, & Leandersson K. (2014). WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells. Molecular Cancer, 13, 88.

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