Cells and 100,000 g cell pellets were lysed in RIPA buffer (10mM Tris-HCL, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140mM NaCl) with 1mM PMSF for 15min on ice. Protein was quantified using a Pierce BCA Protein Assay (Thermo Scientific). Lysates were resolved on a 4%–12% BisTris Bolt mini gel (Invitrogen) and transferred to nitrocellulose membrane. Membranes were blocked with 5% powdered milk or 5% BSA and washed with TBS-T (0.1% Tween-20, 137mM NaCl, 20mM Tris Base). Primary antibodies were used at manufacturer recommended dilutions and included anti-Alix (Abcam, EPR15314 ab186429, RRID: AB_2754981), anti-Tsg101 (Abcam, ab30871, RRID: AB_2208084), anti-CD63 (Abcam, EPR21151, ab217345, RRID: AB_2754982), anti-Syntenin (Abcam, ab19903, RRID: AB_445200), anti-Arf6 (Abcam, ab77581, RRID: AB_2058475), and anti-CD9 (Santa Cruz, KMC8.8, sc-18869, RRID: AB_2076043). Secondary HRP antibodies were goat anti-rabbit (Invitrogen, A16110, RRID: AB_2534782) and donkey anti-rat (Invitrogen, A18745, RRID: AB_2535522), and blots detected with Pierce ELC Western Substrate (Thermo) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) on an AI600 imager (GE Healthcare). Bands were quantified using ImageJ, RRID:SCR_003070.
Epr21151
Manufactured by Abcam
Sourced in United States
EPR21151 is a recombinant monoclonal antibody that recognizes Human Endothelin 1. This antibody is suitable for use in various immunological techniques, including Western Blot, ELISA, and Immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Ab30871, by Abcam (2 mentions)
Ab19903, by Abcam (2 mentions)
Ab77581, by Santa Cruz Biotechnology (2 mentions)
Ab217345, by Abcam (2 mentions)
A16110, by Thermo Fisher Scientific (2 mentions)
Bistris bolt mini gel, by Thermo Fisher Scientific (2 mentions)
Epr15314 ab186429, by Abcam (2 mentions)
A18745, by Thermo Fisher Scientific (2 mentions)
Pierce elc western substrate, by Thermo Fisher Scientific (2 mentions)
Ai600 imager, by GE Healthcare (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Novex bolt transfer buffer, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Chemidoc image system, by Bio-Rad (1 mentions)
Ab125011, by Abcam (1 mentions)
Ice cold lysis buffer, by Roche (1 mentions)
4 protocols using epr21151
1
Western Blot Analysis of Extracellular Vesicles
2
Exosomal CD63 Expression Analysis
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Western blot analysis was performed to analyse CD63 (exosome marker) expression. Cell Lysis Buffer (Cell Signaling Technology) was added to MC4 exosomes (MC4exo) in PBS, sonicated and total protein concentration measured via Bio‐Rad Protein Assay Dye Reagent Concentrate. Total protein (10 μg) was resolved by SDS‐PAGE and transferred to a PVDF membrane in a Mini Trans‐Blot Cell (Bio‐Rad Laboratories Inc.) at 100 V for 2 h in Novex BoltTM transfer buffer (Thermo Fisher Scientific). After blocking with 5% skim milk in Tris‐buffered saline with Tween‐20 (TBS‐T) at RT (1 hr), the PVDF membrane was incubated with anti‐CD63 rabbit antibody [EPR21151] (1:5000; Abcam) overnight at 4°C and probed with anti‐rabbit IgG, HRP‐linked antibody (1:2000; Cell Signaling Technology). Chemiluminescent detection was performed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) in a ChemiDoc image system (Bio‐Rad Laboratories Inc.).
3
Western Blot Analysis of Extracellular Vesicles
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Cells and 100,000 g cell pellets were lysed in RIPA buffer (10mM Tris-HCL, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140mM NaCl) with 1mM PMSF for 15min on ice. Protein was quantified using a Pierce BCA Protein Assay (Thermo Scientific). Lysates were resolved on a 4%–12% BisTris Bolt mini gel (Invitrogen) and transferred to nitrocellulose membrane. Membranes were blocked with 5% powdered milk or 5% BSA and washed with TBS-T (0.1% Tween-20, 137mM NaCl, 20mM Tris Base). Primary antibodies were used at manufacturer recommended dilutions and included anti-Alix (Abcam, EPR15314 ab186429, RRID: AB_2754981), anti-Tsg101 (Abcam, ab30871, RRID: AB_2208084), anti-CD63 (Abcam, EPR21151, ab217345, RRID: AB_2754982), anti-Syntenin (Abcam, ab19903, RRID: AB_445200), anti-Arf6 (Abcam, ab77581, RRID: AB_2058475), and anti-CD9 (Santa Cruz, KMC8.8, sc-18869, RRID: AB_2076043). Secondary HRP antibodies were goat anti-rabbit (Invitrogen, A16110, RRID: AB_2534782) and donkey anti-rat (Invitrogen, A18745, RRID: AB_2535522), and blots detected with Pierce ELC Western Substrate (Thermo) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) on an AI600 imager (GE Healthcare). Bands were quantified using ImageJ, RRID:SCR_003070.
4
Protein Extraction and Western Blot Analysis
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Protein homogenates from A549 cells and H2170 cells were extracted as previously described. Brie y, the cells were lysed for 20 min on ice in ice-cold lysis buffer (Roche). The lysates were centrifuged at 12,000 × g for 20 min at 4•C to obtain a clear lysate. The protein content of each sample was determined using the BCA Protein Assay Kit (Thermo Scienti c). Then, equal amounts of proteins(12μg/lane) were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedi uoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA).The membranes were blocked in 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline-0.1% Tween) at 25 °C for 3 h and then incubated with the following primary antibodies: β-actin (1:800, Abcam, EPR16769), RAB35 (1:700, Abcam, ab152138), TSG101 (1:1500, Abcam, ab125011), CD63 (1:1000, Abcam, EPR21151), HSP70
(1:1000, Abcam, EPR16892). The bands were visualized using horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:2,000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA).
(1:1000, Abcam, EPR16892). The bands were visualized using horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:2,000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA).
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