LPS, GW4869 and PKH-67 were purchased from Sigma-Aldrich. The following antibodies were used: anti-CD68, anti-CD9, anti-CD63, anti-PTEN, anti-Alix, anti-Akt, anti-p-Akt, goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) (Abcam), anti-p65, and anti-p-p65(Beyotime); miR-92a-3p mimic, mir-92a-3p inhibitor, control RNAs and the primers for miRNAs were all purchased from Guangzhou RiboBio.
Anti alix
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Alix is a primary antibody that binds to the Alix protein. Alix is a cytoplasmic protein involved in various cellular processes, including endosomal trafficking, apoptosis, and viral budding. The antibody can be used to detect and study the Alix protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd63, by Abcam (20 mentions)
Anti tsg101, by Abcam (17 mentions)
Anti cd9, by Abcam (12 mentions)
Anti cd63, by Santa Cruz Biotechnology (5 mentions)
Anti cd81, by Abcam (5 mentions)
Anti akt, by Abcam (4 mentions)
Anti calnexin, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti cd63, by System Biosciences (4 mentions)
Anti p akt, by Abcam (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Anti β actin, by Abcam (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti β actin, by Proteintech (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Anti pten, by Abcam (2 mentions)
Pkh67, by Merck Group (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti ki67, by Abcam (2 mentions)
40 protocols using anti alix
1
Exosome Regulation by miR-92a-3p
2
Comprehensive Protein Expression Profiling of Extracellular Vesicles
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Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.
3
Exosome Protein Profiling by Western Blot
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Exosomes and cell lysates were spotted on a nitrocellulose membrane (Thermo Fisher Scientific, UK) (0.5 µg protein in 40 µL for all samples – 10 µL at a time, dried under a nitrogen stream before addition of the next 10 µL on the same spot). The membrane was blocked with 3% milk (w/v) prepared in TBS-T (TBS pH 7.6 containing 0.1% Tween-20) for 1 h at RT. The membrane was then incubated with primary rabbit anti-human/mouse antibodies: anti-Alix (monoclonal, clone 3A9, ab117600, Abcam, UK), anti-TSG101 (polyclonal, 14497-1-AP, ProteinTech, UK), anti-CANX (polyclonal, 10427-2-AP, ProteinTech, UK) and anti-GAPDH (monoclonal, clone 14C10, #2118, Cell Signalling Technology, UK) antibodies (1:1000 in 3% milk), overnight at 4°C. The membrane was washed three times with TBS-T, followed by incubation with goat anti-rabbit secondary antibody (polyclonal, ab6721, 1:1000 in 3% milk) for 1 h at RT. The membrane was then washed again as above, and SuperSignal™ West Femto Maximum Sensitivity ECL substrate (Thermo Fisher Scientific, UK) was added to the membrane (50 µL per sample spot). The membrane was incubated with the substrate for 2 min at RT, and then imaged using the Gel Doc™ system (Bio-Rad, USA) under the “Intense Bands” setting. The image obtained was analysed using the Image Lab™ software (Bio-Rad, USA).
4
Extracellular Vesicle Protein Profiling
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Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), which were treated with 5% milk in PBS at room temperature for 1 h. Then the membranes were incubated at 4 °C overnight with anti-TSP1 (Abcam, Cambridge, UK), anti-CD63 (Abcam), anti-TSG101 (Abcam), anti-Calnexin (Abcam), anti-Alix (Abcam), or anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) antibodies. Then the membranes were treated with fluorescent dye-conjugated secondary antibody and visualized using Odyssey CLx (LI-COR, Lincoln, NE, USA). The strength of the signal for each protein was determined based on the corresponding band intensity of the scanned image.
5
Western Blot Analysis of Extracellular Vesicle Markers
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The Western blot was conducted following routine procedures, as described previously (20 (link)). Briefly, proteins were extracted using radioimmunoprecipitation buffer containing protease inhibitor cocktail (Abcam). Protein concentration was measured by BCA assay kit (Abcam). The 20 µg of protein were loaded on SDS-PAGE gel and then subjected to transfer. The primary antibodies used in the study included: anti-CD63 (1:1,000; Abcam), anti-β actin (1:2,000; Abcam), anti-CD9 (1:1,000; Abcam), anti-Alix (1:1,000; Abcam), anti-TLR4 (1:1,000; Abcam), anti-phospho-p65 (1:1,000; Abcam), anti-p65 (1:1,000; Abcam).
6
Extracellular Vesicle Protein Profiling
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Western blot analysis was performed on 100 μL of plasma samples incubated overnight with 5 μL of MACSPlex detection beads at 10°C at 800 rpm. The next day, the unbounded fraction was discarded, and samples were lysed with radioimmunoprecipitation assay buffer. Total proteins were separated on a gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis 4%–12% gel and transferred onto polyvinylidene difluoride membrane. The blot was incubated with the following primary antibodies: anti-Alix (rabbit polyclonal; Abcam, Cambridge UK, 1:1,000); anti–tumor susceptibility gene 101 (TSG101) (rabbit polyclonal; Abcam, 1:1,000); anti-CD81 (mouse monoclonal, Thermo Fisher Scientific, Waltham, MA, 1:300); anti–apolipoprotein A1 (APOA1) (rabbit polyclonal; Abcam, 1:300); and anti-GRP94 (rabbit polyclonal; Abcam, 1:500).
7
Characterization of Extracellular Vesicles
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EV sample was concentrated using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany), and protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Scientific). Concentrated sEV preparations and lysate of HCT116 cells were lysed in Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific), heated for 5 min at 95 °C, and subjected to electrophoresis using 10% SDS-PAGE. Proteins were transferred to an Immobilon-P PVDF Membrane (Merck Millipore) and the excess protein binding sites on the membrane were saturated with 5% bovine serum albumin blocking buffer (1 × TBS, 0.1% Tween-20) for 1 h. The membrane was incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-CD81 (1:250, mouse, catalogue number sc166029), anti-CD63 (1:300, mouse, sc5275) from Santa Cruz Biotechnology, anti-Alix (1:20000, rabbit, ab186429) from Abcam, anti-TSG101 (1:200, mouse, 612696) from BD Biosciences, and anti-Calnexin (1:1000, rabbit, 2679) from Cell Signaling. After incubation, the membrane was washed three times with 5% TBS-Tween and then, incubated with peroxidase-labelled secondary antibody (Santa Cruz Biotechnology) for one hour. After three washes, immobilized proteins were detected utilizing Clarity Western ECL Substrate (Bio-Rad) and the UVITEC chemiluminescence imager (UVITEC Cambridge, UK).
8
Extracellular Vesicle Protein Profiling
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Cell or tissue samples were lysed using RIPA reagent supplemented with PMSF (Roche), protease inhibitor (Roche), and phosphatase inhibitor (BestBio). Proteins were separated by SDS‐PAGE and blotted onto PVDF membranes. The primary Abs anti‐ALIX, anti‐TSG101, anti‐CD63, anti‐CD81, anti‐Calnexin, anti‐EphB2, anti‐p‐PI3K, anti‐PI3K, anti‐p‐AKT, anti‐AKT, and anti‐GAPDH were purchased from Abcam. Protein bands could be imaged after development of the blots using the chemiluminescence kit (Vazyme).
9
Exosome Protein Marker Analysis
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The protein samples were extracted and separated by 10% SDS-PAGE gel, and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skimmed milk and incubated overnight, using the following main detection antibodies at 4°C: anti-CD63 (1:1,000; Abcam, UK), anti-TSG101 (1:1,000; Abcam), anti-Alix (1:1,000; Abcam), anti-Hsp90 (1:1,000; Abcam), anti-GRP94 (1:1,000; Abcam), anti-Cytochrome (1:1,000; Abcam), anti-p-pi3k (1:1,000; Abcam), anti-pi3k (1:1,000; Abcam), anti-p-akt (1:1,000; Abcam), anti-akt (1:1,000; Abcam), anti-E-cadherin (1:1,000; Abcam), anti-vimentin (1:1,000; Abcam), or anti-b-actin (1:5,000; Proteintech, USA). We washed 3 times with TBS-T and the membranes were cultured with the secondary antibody at 24°C for 1 h. The western blots were pictured using an ECL Reagent (Pierce, USA) and the density was verified using ImageJ software (NIH, USA).
10
Protein Separation and Analysis
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The newly added protease inhibitor and the lysis buffer (SDS) were applied for protein separation. Subsequent to separation through SDS-PAGE, the lysates were transferred onto a polyvinylidene fluoride membrane acquired from Roche. Then the membrane was blocked with 2% BSA, followed by incubation with anti-CD63 and anti-TSG 101 from Santa Cruz, and anti-Alix, ZEB1 antibody, MTOR antibody, DNMT3A antibody and GAPDH antibody from Abcam at 4°C overnight. Then the membrane underwent incubation with the proper secondary antibodies, and the protein expression in cells was measured with GAPDH as an internal reference.
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