Serum sEV and human 293T cells (Takara catalog 632180) were resuspended in RIPA lysis buffer (MilliporeSigma) containing 0.1% SDS and protease and phosphatase inhibitors (Biotool) and incubated on ice for 30 minutes prior to centrifugation at 12,000g for 10 minutes. Supernatants were used for Western blotting in SDS-PAGE electrophoresis. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). The following antibodies were used: TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (ab68418, Abcam), and calnexin (ab22595, Abcam).
Ab22595
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab22595 is a monoclonal antibody that targets the Histone H3 (tri methyl K9) protein. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab92726, by Abcam (18 mentions)
Ab125011, by Abcam (17 mentions)
Bca kit, by Thermo Fisher Scientific (10 mentions)
Ab134045, by Abcam (8 mentions)
Ab68418, by Abcam (6 mentions)
Ab9485, by Abcam (6 mentions)
Ab217345, by Abcam (5 mentions)
Ab109201, by Abcam (5 mentions)
Ab223052, by Abcam (5 mentions)
Ab216130, by Abcam (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Ab6721, by Abcam (4 mentions)
Ripa lysis buffer, by Merck Group (4 mentions)
Ab79559, by Abcam (3 mentions)
Ab30871, by Abcam (3 mentions)
Ab236630, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab52649, by Abcam (3 mentions)
A5271, by ABclonal (3 mentions)
99 protocols using ab22595
1
Extracellular Vesicle Protein Analysis
2
Exosome Marker Protein and GFP Detection
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For the detection of EV (exosome) marker proteins and GFP, isolated EVs or lysate of CD63‐GFP‐HeLa cells was added to the SDS sample buffer. The boiled samples were separated via 10% SDS/PAGE, transferred onto polyvinylidene fluoride membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti‐CD63 antibody (TS63; Abcam, Cambridge, UK), anti‐ALIX antibody (ab88388; Abcam), anti‐calnexin antibody (ab22595; Abcam), and anti‐GFP antibody‐ChIP grade (ab290; Abcam). A secondary antibody labeled with horseradish peroxidase [anti‐mouse IgG HRP NA931V (GE Healthcare) and goat anti‐rabbit IgG (ab6721, Abcam)] was used, and the immunoreactive species were detected using the Enhanced Chemiluminescence (ECL) Plus Western Blotting Detection System (GE Healthcare) with the Amersham Imager 600 (GE Healthcare) and iBright western blot imaging system (Thermo Fisher Scientific Inc.).
3
Immunofluorescence and Transmission Electron Microscopy of Cellular Structures
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For immunofluorescence, cells were fixed in 4% PFA in 1× PBS for 15 to 20 minutes and permeabilized for 5 minutes with 0.1% Triton X-100 in PBS. Cells were blocked for 30 minutes with 5% goat serum and then incubated with primary antibodies (CD63 1:200, ab59479, Abcam; GluN1 1:500, MAB363, Merk & Co, Kenilworth, New Jersey, United States or Calnexin 1:1000, ab22595, Abcam) at 4°C overnight. After washing, cells were incubated with 2.5 μg mL
−1Dylight 488/594-conjugated secondary antibodies (ab96931 and ab96885, respectively; both from Abcam) for 3 hours. Giemsa staining was performed using the Cytopro autostainer (ELITech, Paris, France). Brightfield and immunofluorescence microscopy was conducted using an Eclipse Ni-E microscope (Nikon, Tokyo, Japan). Hoffman and phase contrast images were taken on an Eclipse Ti microscope (Nikon).
Transmission electron microscopy was done as previously described.
18 (link)
Briefly, cells were fixed with 0.2% glutaraldehyde and 2% PFA in White's saline. Sections were counterstained with uranyl acetate and examined with a Tecnai G2 Spirit Twin transmission electron microscope (FEI Company, Hillsboro, Oregon, United States).
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For immunofluorescence, cells were fixed in 4% PFA in 1× PBS for 15 to 20 minutes and permeabilized for 5 minutes with 0.1% Triton X-100 in PBS. Cells were blocked for 30 minutes with 5% goat serum and then incubated with primary antibodies (CD63 1:200, ab59479, Abcam; GluN1 1:500, MAB363, Merk & Co, Kenilworth, New Jersey, United States or Calnexin 1:1000, ab22595, Abcam) at 4°C overnight. After washing, cells were incubated with 2.5 μg mL
−1Dylight 488/594-conjugated secondary antibodies (ab96931 and ab96885, respectively; both from Abcam) for 3 hours. Giemsa staining was performed using the Cytopro autostainer (ELITech, Paris, France). Brightfield and immunofluorescence microscopy was conducted using an Eclipse Ni-E microscope (Nikon, Tokyo, Japan). Hoffman and phase contrast images were taken on an Eclipse Ti microscope (Nikon).
Transmission electron microscopy was done as previously described.
18 (link)
Briefly, cells were fixed with 0.2% glutaraldehyde and 2% PFA in White's saline. Sections were counterstained with uranyl acetate and examined with a Tecnai G2 Spirit Twin transmission electron microscope (FEI Company, Hillsboro, Oregon, United States).
4
Comprehensive Protein Expression Analysis
5
Protein Quantification and Immunodetection
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Protein quantification was performed using a Pierce bicinchoninic acid protein assay kit (ThermoFisher), with electrophoresis using SDS-PAGE and immunodetection using calnexin (Abcam, ab22595) and ATP5A (Abcam, ab14748) followed by fluorescent DyLight secondary antibodies (Invitrogen, SA5-10036 and 35519) and visualisation using a CLx imaging system scanner (LI-COR Bioscience), using Geneflow BLUeye protein ladder (245kDa) ladder (Supplementary Fig. 1 ).
6
Exosome Isolation and Characterization
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After culturing in FBS-free media for 48 h (to eliminate interference from FBS-secreted exosomes), an ExoQuick-TC kit (EXOTC50A-1, EXOQ20A-1, System Biosciences, Palo Alto, CA, USA) was used to extract exosomes following the protocol provided by the manufacturer. This was followed by characterization of the isolated exosomes using the following biomarkers: HSP70 (Abcam, Cambridge, UK, ab133586, 1:2,000), TSG101 (Abcam, Cambridge, UK, ab133586, 1:1,000), and calnexin (Abcam, Cambridge, UK, ab22595, 1:1,000). The shapes and sizes of exosomes were analyzed using TEM (FEI, Hillsboro, OR, USA) and DLS (Malvern Instruments, Worcestershire, UK), respectively.
7
Immunofluorescence Staining and Confocal Microscopy
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Cells grown on coverslips were washed gently with phosphate-buffered saline (PBS) and fixed in 4% PFA, followed by permeabilization with 0.3% Triton X-100 in PBS. The cells were then blocked for 1 h in 5% goat serum, followed by incubation overnight with primary antibodies at 4°C. Mouse anti-FLAG antibody (Beyotime biotechnology, AF519-1, 1:500 dilution), rabbit anti-FLAG antibody (Beyotime biotechnology, AF0036, 1:500 dilution), anti-GJB1 (Cx32) antibody (Santa Cruz Biotechnology,#10519, 1:150 dilution), anti-Calnexin antibody (Abcam, ab22595, 1:400 dilution), anti-GM130 antibody (Sigma-aldrich, 610822, 1:400 dilution), and anti-G3BP1 antibody (Beyotime biotechnology, AF0039, 1:150 dilution) were used. Secondary antibodies were Alexa Fluor (488, 555)-labeled goat anti-mouse and goat anti-rabbit antibodies (Thermo Fisher, 1:500 dilution). DAPI was used to visualize the nuclei. Fluorescence images were captured with a 20 × or 63 × objective on a Zeiss LSM780 laser scanning confocal microscope.
8
Immunoblot Analysis of Alzheimer's Markers
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The following antibodies were used for Immunoblot: mouse anti presenilin 1, dilution 1:1,000 (MAB5232; Merck Millipore); rabbit anti-Amyloid Precursor Protein, C-terminal antibody, dilution 1:1,000 (A8717; Sigma-Aldrich); rabbit anti-neuregulin 1, dilution 1:1,000 (NBP2-19588; Novus Biologicals); mouse anti-PSD95, dilution 1:1,000 (610495; BD Biosciences); mouse anti-tau5, dilution 1:1,000 (AHB0042; Thermo Fisher Scientific/Invitrogen); rabbit anti-tau-P Ser 396, dilution 1:1,000 (44752G; Life Technologies); rabbit anti-tau-P Ser 404, dilution 1:1,000 (44758G; Life Technologies); mouse anti-IRS1, dilution 1:1,000 (BD Biosciences); rabbit anti-IRS1-P Ser307, dilution 1:500 (ab1194; Abcam); rabbit anti-synaptophysin, dilution 1:1,000 (ab-14692; Abcam); rabbit anti-calnexin, dilution 1:10,000 (ab22595; Abcam); mouse anti-β-actin, dilution 1:10,000 (A5441; Sigma-Aldrich).
9
Characterization of Extracellular Vesicles
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The prepared EVs were fixed with 2% glutaraldehyde (Sigma, USA). After 30 min, 10 µL of fixed samples was pipetted onto copper grids with carbon-coated formvar film and incubated for 10 min. Grids were washed three times with ddH2O and the excess liquid was removed by blotting. Micrographs of sEV were obtained with a Transmission electron microscopy (JEM-2100F electron microscope). For particle size analysis, dynamic optical diffraction was used to fit the curve.
Western blots were performed in the standard fashion using Mini-PROTEAN 4-20% SDS-PAGE gels and the Trans-Blot Turbo Transfer System (Bio-Rad). The following antibodies were used: anti-CD9 mouse antibody (Abcam, [MEM-61] (ab2215)), anti-CD81 mouse antibody (Abcam, [M38] (ab79559)), anti-calnexin rabbit antibody (Abcam, (ab22595)), anti-RGD rabbit antibody (Abcam, (ab224465)).
Western blots were performed in the standard fashion using Mini-PROTEAN 4-20% SDS-PAGE gels and the Trans-Blot Turbo Transfer System (Bio-Rad). The following antibodies were used: anti-CD9 mouse antibody (Abcam, [MEM-61] (ab2215)), anti-CD81 mouse antibody (Abcam, [M38] (ab79559)), anti-calnexin rabbit antibody (Abcam, (ab22595)), anti-RGD rabbit antibody (Abcam, (ab224465)).
10
Protein Expression Analysis in Liver Microsomes
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Hepatic microsomes or liver lysates (10–30 µg protein) were separated on a 10% SDS polyacrylamide gel (Bio-Rad, Hercules, CA, USA) by electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 at room temperature for an hour, followed by incubation with primary antibodies against β-actin (Sigma, A1978, 1:7500), calnexin (Abcam, ab22595, 1:3000), CYP2E1 (Abcam, ab28146, 1:5000), CYP1A2 (Chemicon, Mab10035, 1:5000), voltage-dependent anion channel (VDAC; ThermoFisher Scientific, PA1-954A, 1:3000), α-tubulin (Calbiochem, CP06, 1:10000), JNK (Cell Signaling, 9252, 1:1000), or pJNK (Cell Signaling, 9251, 1:1000) overnight at 4°C. On the next day, the membrane was incubated with a secondary antibody (1:10000) for one hour, and the proteins were detected by a chemiluminescent ECL reagent (Pierce, Rockford, IL, USA). The intensity of protein bands was quantified using Image J.
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