Cell lysate was collected in 8 M urea with 2.5% SDS. Protein concentration was measured with Qubit Protein Assay (Thermo Fisher), with 30 μg of cell lysate and 15 μg of exosome protein loaded. Protein was denatured in 4× NuPAGE LDS sample buffer with 62.5 mM DTT for 10 min at 70 °C. Membranes were blocked with 5% milk in TBS with 0.1% Tween (TBST) and incubated with rabbit anti-SLC19A1 (Boster Biological Technology PB9504, 1:1000), rabbit anti-SLC38A2 (Sigma Aldrich SAB4502246, 1:1000), mouse anti-CD81 (Santa Cruz Biotechnology sc-166029, 1:1000), or rabbit anti-vinculin (Abcam ab129002, 1:1000) for 1 h at room temperature or overnight at 4 °C. Membranes were incubated in anti-rabbit HRP (Abcam ab16284, 1:1000) or anti-mouse HRP (R&D Systems HAF007, 1:1000) for 1 h at room temperature and developed using West-Q Pico ECL solution (GenDEPOT).
Sc 166029
Manufactured by Abcam
Sc-166029 is a primary antibody produced in rabbit. It targets the protein DHPR. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ab16284, by R&D Systems (3 mentions)
Haf007, by R&D Systems (3 mentions)
Ab133267, by Abcam (2 mentions)
Qubit protein assay, by Thermo Fisher Scientific (1 mentions)
Ab129002, by Abcam (1 mentions)
West q pico ecl solution, by GenDEPOT (1 mentions)
H 7650, by Hitachi (1 mentions)
Zetaview pmx 110, by Particle Metrix (1 mentions)
Ab125011, by Abcam (1 mentions)
4 protocols using sc 166029
1
Exosomal Protein Profiling by Western Blot
2
Hypoxia and Gemcitabine Modulate Extracellular Vesicles
3
Characterization of Adipose-Derived Extracellular Vesicles
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The size of ADSCs‐exo was measured by nanoparticle tracking analysis (NTA) with ZetaView PMX110 (Particle Metrix). The morphology of ADSCs‐exo was analysed by transmission electron microscopy (TEM) H‐7650 (Hitachi). Western blot was performed using anti‐CD9 (Abcam, ab92726, 1:2000 dilution), anti‐CD81 (Santa, sc‐166029, 1:500 dilution) and anti‐TSG 101 (Abcam, ab125011, 1:5000 dilution).
4
Extracellular Vesicle Profiling in Hypoxia/Gemcitabine
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MIA PaCa-2 cells (triplicates) were exposed to hypoxia (1%O2)/normoxia (21% O2) or to gemcitabine (1 µM)/vehicle (cell culture grade water) for 72 hours and cultured in RPMI medium supplemented with EVs-depleted FBS. EVs were collected as previously described and pellets were resuspended in 100 µL of PBS 1×. For western blot, 20 µL of EV preparation from each condition was loaded into the gel. CD81 (1:5000 Santa Cruz Cat# sc-166029, RRID:AB_2275892), syntenin-1 (1:5000, Abcam Cat# ab133267, RRID:AB_11160262) and CD9 (1:1000, Abcam Cat# ab263019) were used to detect EVs.
Antirabbit-HRP 1:5000 (CST Cat# 7074S, RRID:AB_2099233) was used for syntenin-1, antirabbit-HRP 1:2000 (Abcam, Cat# ab16284, RRID:AB_955387) was for CD9 and anti-mouse-HRP (1:2000 R&D Cat# HAF007, RRID:AB_357234) for CD81.
Antirabbit-HRP 1:5000 (CST Cat# 7074S, RRID:AB_2099233) was used for syntenin-1, antirabbit-HRP 1:2000 (Abcam, Cat# ab16284, RRID:AB_955387) was for CD9 and anti-mouse-HRP (1:2000 R&D Cat# HAF007, RRID:AB_357234) for CD81.
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