Exosomes lysed with M-PER (10 μg) were separated through SDS-PAGE, then transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, United States). After blocking with bullet blocking one (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with rabbit polyclonal anti-AKR1B1 (1:500, sc-33219, Santa Cruz Biotechnology, Dallas, TX, United States), goat polyclonal anti-CAPG (1:100, sc-33084, Santa Cruz Biotechnology), rabbit polyclonal anti-HSP70 antibody (1:2,000, EXOAB-HSP70A-1, System Biosciences), rabbit polyclonal anti-CD63 (1:1,000, EXOAB-CD63A-1, System Biosciences), or rabbit polyclonal anti-RAB5 (ab13253, 1:1,000, Abcam, Tokyo, Japan). Immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, United States) after incubation with horseradish peroxidase-labeled goat anti-rabbit IgG or horse anti-goat IgG (1:5,000, Vector Laboratories, Burlingame, CA, United States). Signals were detected using C-DiGit Blot Scanner (LI-COR; Kusama et al., 2018a (link)). Total proteins were stained with colloidal gold total protein stain solution according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, United States).
Exoab cd63a 1
Manufactured by System Biosciences
Sourced in United States, Japan, United Kingdom
The EXOAB-CD63A-1 is an exosome isolation and enrichment kit. It utilizes CD63-specific antibodies to capture and purify exosomes from cell culture media or biological fluids.
Automatically generated - may contain errors
Lab products found in correlation
Exoab cd9a 1, by System Biosciences (45 mentions)
Exoab hsp70a 1, by System Biosciences (12 mentions)
Exoab tsg101 1, by System Biosciences (9 mentions)
Pvdf membrane, by Bio-Rad (7 mentions)
C digit blot scanner, by LI COR (6 mentions)
Exoab cd81a 1, by System Biosciences (6 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (6 mentions)
Sc 6037, by Santa Cruz Biotechnology (6 mentions)
Protease inhibitor, by Thermo Fisher Scientific (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Yt0613, by Immunoway (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab13253, by Abcam (3 mentions)
Colloidal gold total protein stain solution, by Bio-Rad (3 mentions)
Bullet blocking one, by Nacalai Tesque (3 mentions)
Sc 33219, by Santa Cruz Biotechnology (3 mentions)
30 protocols using exoab cd63a 1
1
Exosomal Protein Expression Analysis
2
Characterization of Adipose-Derived Stem Cell Extracellular Vesicles
3
Extracellular Vesicle Protein Profiling
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EV marker and immuno-related proteins in lysates from EVs and EECs were detected through the use of SDS-PAGE. Separated proteins transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) were blocked with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), and the membranes were incubated with the following antibodies; rabbit polyclonal anti-human CD63 antibody (1:2000, EXOAB-CD63A-1, System Biosciences), rabbit polyclonal anti-human HSP70 antibody (1:2000, EXOAB- HSP70A-1, System Biosciences), rabbit polyclonal anti-bovine CTSC (1:2000, ab182904, abcam, Tokyo, Japan), rabbit polyclonal anti-pocine IL6 (1:2000, ab193853, abcam), or rabbit polyclonal anti-human ACTB (1:5000, ab1801, abcam). The membranes were then incubated with secondary antibody, horseradish peroxidase labeled goat anti-rabbit IgG (1:5000, Vector Laboratories, Burlingame, CA, USA), and immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, USA). Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2)37 (link).
4
Western Blot Analysis of Protein Modifications
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SDS-PAGE was performed using 10% (w/v) gels and the proteins were transferred onto nitrocellulose membranes (0.2 µm pore size) by overnight blotting performed at 150 Vh. Total AnxA2 or its Tyr23-phosphorylated form were detected using monoclonal antibodies directed against AnxA2 (610069, BD Biosciences, 1:1000) or pTyr23AnxA2 (sc-135753, Santa Cruz Biotechnologies, 1:200) (Spijkers-Hagelstein et al., 2013 (link)). CD63 and T-cadherin were detected using rabbit polyclonal antibodies (EXOAB-CD63A-1, System Biosciences, 1:500 and sc-7940, Santa Cruz, 1:1000); ubiquitin was detected using mouse monoclonal antibodies [13-1600 (Ubi-1), Invitrogen, 1:1000]. Activated Src (pTyr416 Src), Src and fibrillarin were detected by monoclonal rabbit antibodies from Cell Signaling at a 1:1000 dilution (D49G4, 32G6 and C13C3, respectively). Subsequently, HRP-labelled secondary anti-rabbit or anti-mouse antibodies (170-6515, Bio-Rad, 1:5000 and 115-035-174, Jackson ImmunoResearch Laboratories, 1:5000) were used. The reactive protein bands were visualised using the Supersignal West Pico- or Femto Chemiluminescent Substrate kits (Pierce).
5
Exosome Protein Analysis by Western Blot
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1:100 diluted phosphatase inhibitor cocktails I&II (Millipore Sigma) and 1:100 protease inhibitors (Thermo Fisher) were added to RIPA buffer to decrease sample degradation. Protein was quantified using a Pierce BCA kit and 10 μg of each sample was separated with a 12% SDS-PAGE gel and transferred onto PVDF membranes. Membranes were blocked in 5% BSA, 1X TBS and 0.1% Tween-20 for 1 hour. Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. We used anti-CD63 (1:1000 dilution, EXOAB-Cd63A-1, System Biosciences), anti-TSG101 (1:1000 dilution, ab83, Abcam), anti-CAMK1D (1:800 dilution, #3365, Cell Signaling) and loading control anti-Histone H3 (1:4000, # 4499 Cell Signaling) followed by HRP conjugated anti-rabbit (1:2000, #7074, Cell Signaling). Detection was done using an Amersham ECL kit and hyperfilm (General Electric). Western images were quantified using the ‘gel analysis’ function in ImageJ (http://imagej.nih.gov ).
6
Exosome Protein Profiling by Western Blot
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Cells and exosomes were lysed in ice-cold cell lysis buffer containing 50 mM Tris/HCl (PH7.5), 2 mM EDTA, 150 mM NaCl, 1 % (v/v) Triton X-100, complete™ proteinase and phosphatase inhibitors (Roche), as described previously [5 (link)]. Lysates were loaded and subjected to SDS–polyacrylamide (4–20 %) electrophoresis and electroblotted onto PVDF membranes. After blocking in 5 % non-fat dry milk, membranes were incubated with mouse anti-polyglutamine (1C2, Cat. No. MAB1574, Millipore), mouse anti-total huntingtin (4C8, Cat. No. MAB2166, Millipore) and mouse anti-mutant huntingtin (EM48; 1:1000) antibodies. Exosomes were identified with the antibodies raised against CD9 (1:1000, Cat. No. EXOAB-CD9A-1, Systems Biosciences), CD63 (1:1000, Cat. No. EXOAB-CD63A-1, Systems Biosciences), CD81 (1:1000, Cat. No. EXOAB-CD81A-1, Systems Biosciences) and HSP70 (1:1000, Cat. No. EXOAB-Hsp70A-1, Systems Biosciences). Membranes were incubated with the appropriate secondary antibody (goat anti-mouse or anti-rabbit) according to the manufacturer’s instructions, followed by the addition of the chemiluminescent detection reagent (Millipore). Bands were visualized using a ChemiDoc™ XRS + system (Bio-Rad) and quantified using the image lab software (Bio-Rad).
7
Detecting EV Proteins by Western Blot
8
Exosomal Protein Characterization Protocol
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Exosomes were lysed in cold RIPA buffer with PMSF. The total protein concentration was determined by BCA assay. Protein samples were mixed with 5× loading buffer and separated by 10% SDS polyacrylamide gels. Then the proteins were transferred to PVDF membranes (Millipore, NJ, United States) and incubated with 5% non-fat milk at room temperature for 1 h. Exosomes were identified using 2 positive markers including CD9 (1:1,000; EXOAB-CD9A-1, System Biosciences) and CD63 (1:1,000; EXOAB-CD63A-1, System Biosciences). Calnexin (1:1,000; YT0613, Immunoway Biotechnology) was used as a negative marker for exosomes.
9
Western Blotting of Exosomal CD63
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For Western blotting, 4 μL of Type A exosomes were mixed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), then with Laemmli sample buffer containing β-mercaptoethanol (Bio-Rad, Oxford, UK), denatured at 95 °C for 5 min, loaded to Mini-PROTEANR TGX™ 10% gradient SDS-PAGE gels (Bio-Rad, Oxford, UK), and blotted on Immobilon-P membranes (Millipore, Bedford, MA, USA). Blocking (1 h, RT) and antibody incubations (the primary antibodies overnight at 4 °C; the secondary antibodies 1.5 h at RT) were performed in 5% non-fat powdered milk (Valio, Helsinki, Finland) in TBS and in TBS with 0.1% Tween-20, respectively. The anti-human CD63 antibody (EXOAB-CD63A-1; 1:1000) was purchased from System Biosciences (Palo Alto, USA), and the anti-bovine CD63 antibody (abx021473; 1:500) from Abbexa (Cambridge, UK). For detection, horseradish (HRP)-conjugated secondary antibodies (HRP-conjugated rabbit anti-mouse IgG, 1:20,000; HRP-conjugated goat anti-human IgG (for detection of T-DM1), 1:20,000, both from Jackson ImmunoResearch; and HRP-conjugated goat anti-rabbit IgG, 1:20,000, System Biosciences) and a 1:2 mixture of SuperSignal™ West Pico Chemiluminescent Substrate and SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA) were used.
10
Proteomic analysis of plasma-derived extracellular vesicles
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The EVs extracted from the plasma samples were re-suspended in 500 μl protein lysis buffer (Beyotime Biotechnology), and 20 μl of each lysed sample was resolved by SDS-PAGE (10% w/v sodium dodecyl sulphate, 30% w/v acrylamide, 1 M Tris-HCl pH 6.8, 10% w/v ammonium persulphate, and 2% TEMED). After transferring to PVDF membranes, the protein bands were probed with anti-CD63 ExoAB (1:1000, Cat. No. ExoAB-CD63A-1, System Biosciences, CA), rabbit anti-TSG101 (clone EPR 7130(B), 1:2000, Cat. No. ab125011, Abcam), rat anti-calnexin (1:500, Cat. No. sc-23954, Santa Cruz), rabbit anti-HSC70 (1:1000, Cat. No. ab51052, Abcam), rabbit anti-FGA (1:2000, Cat. No. ab92572, Abcam), rabbit anti-FN1 (1:1000, Cat. No. ET1702-25, HuaBio), rabbit anti-S100A9 (1:1000, Cat. No. ET1702-73, HuaBio) and rabbit anti-HP (1:1000, Cat. No. ET1703-24, HuaBio) antibodies. The blots were washed with the Tris-Buffered Saline (Boster, USA), 0.1% Tween® 20 Detergent (Sigma, MO, USA) buffer and incubated with IRDye 800-conjugated goat anti-mouse/rabbit IgG (1:1000 or 1:2,500, Li-COR Biosciences) secondary antibodies at room temperature. We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: BR2025GX for plasma and EV170041 for cell lines).
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