Western blot assay was performed following the standard protocol. In brief, after total protein was isolated and quantified, the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Solarbio, Beijing, China) and then transferred onto polyvinylidene difluoride membranes (PVDF; Pall Corporation, New York, NYC, USA). Next, the membranes were blocked in slim milk for 2 h and then incubated with primary antibodies against ATG3 (ab108251; 1:2000; Abcam, Cambridge, MA, USA), LC3Ⅱ/LC3Ⅰ (ab128025; 1:200; Abcam), P62 (ab56416; 1:500; Abcam), B-cell lymphoma-2 (BCL-2; ab196495; 1:1000; Abcam), cleaved caspase-3 (ab49822; 1:500; Abcam), cleaved caspase-9 (ab2324; 1:2000; Abcam), BCL2-Associated X (Bax; ab182733; 1:2000; Abcam) or GAPDH (ab181602; 1:5000; Abcam) at 4°C overnight followed by incubation with HRP-conjugated secondary antibody (D110150; 1:5000; Sangon, Shanghai, China) for 2 h. The protein bands were analyzed using an enhanced chemiluminescence reagent (Beyotime).
Ab108251
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab108251 is a product offered by Abcam. It is a type of lab equipment, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information is not available.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ab128025, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab49822, by Abcam (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab56416, by Abcam (1 mentions)
Sds page, by Solarbio (1 mentions)
Polyvinylidene fluoride (pvdf), by Pall Corporation (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab28233, by Abcam (1 mentions)
Ab133615, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab109201, by Abcam (1 mentions)
Lsm 710 inverted confocal microscope, by Zeiss (1 mentions)
Sc 390351, by Santa Cruz Biotechnology (1 mentions)
Hoescht, by Merck Group (1 mentions)
4 protocols using ab108251
1
Western Blot Analysis of Autophagy and Apoptosis
2
Exosome Characterization in B-ALL
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Western blot was performed to identify exosomes and verify the predicted proteins associated with B-ALL progression. Whole exosomes in each sample were lysed in radioimmunoprecipitation assay lysis buffer and quantified using a bicinchoninic acid protein assay kit (23,227; Thermo Fisher Scientific). Thereafter, 20 μg of protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies supplied by Abcam (Cambridge, UK) against TSG101 (ab125011), CD81 (ab109201), calnexin (ab133615), ATG3 (ab108251), and ADAM17 (ab28233) overnight at 4 °C. The membranes were then incubated for 1 h with a secondary antibody at room temperature. A commercial chemiluminescence kit was used to visualize the immunoreactive blots, which were photographed using a Tanon 5200 camera (Tanon, Shanghai, China).
3
Tissue Fixation and Histological Analysis
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Tissues were fixed in 10% NBF for 24 h before being dehydrated, embedded in paraffin wax, and sectioned (4 μm) using standard procedures as previously described (Zhu et al., 2016) . Sections were dewaxed in xylene and stained with Von Kossa and alizarin red (Sigma) to visualize phosphate and calcium deposition, respectively.
For immunofluorescence analysis, sections were demasked with 10 mM sodium citrate buffer. Endogenous peroxidase and nonspecific antibody binding were blocked before overnight incubation with primary antibodies LC3 (1:300; PM036; MBL International), Runx2 (1:200; Sc-390351; Santa Cruz Biotechnology), or ATG3
(1:300; ab108251; Abcam) at 4°C. The sections were then incubated for 1 h in the dark with Alexa Fluor@488 anti-rabbit antibody (Life Technologies; A11034) and Alexa Fluor@647 goat anti mouse antibody (Life Technologies; A21236). Sections were washed in PBS and stained with Hoescht (1:10,000; Sigma) and then mounted onto slides with Prolong ® Gold Anti-Fade Reagent (Life Technologies).
Fluorescence signal was detected under a Zeiss LSM 710 inverted confocal microscope. Control sections were incubated with nonimmune goat IgG (2 μg IgG/ml) in place of the primary antibody.
For immunofluorescence analysis, sections were demasked with 10 mM sodium citrate buffer. Endogenous peroxidase and nonspecific antibody binding were blocked before overnight incubation with primary antibodies LC3 (1:300; PM036; MBL International), Runx2 (1:200; Sc-390351; Santa Cruz Biotechnology), or ATG3
(1:300; ab108251; Abcam) at 4°C. The sections were then incubated for 1 h in the dark with Alexa Fluor@488 anti-rabbit antibody (Life Technologies; A11034) and Alexa Fluor@647 goat anti mouse antibody (Life Technologies; A21236). Sections were washed in PBS and stained with Hoescht (1:10,000; Sigma) and then mounted onto slides with Prolong ® Gold Anti-Fade Reagent (Life Technologies).
Fluorescence signal was detected under a Zeiss LSM 710 inverted confocal microscope. Control sections were incubated with nonimmune goat IgG (2 μg IgG/ml) in place of the primary antibody.
4
Western Blot Analysis of Osteogenic Markers
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Cells were lysed in radioimmunoprecipitation assay buffer supplemented with Protease Inhibitor Cocktail (Thermo Fisher Scientific) and total protein concentration determined (Thermo Fisher Scientific). Immunoblotting was performed as previously described (Zhu et al., 2016) .
Nitrocellulose membranes were probed overnight at 4°C with primary antibodies (1:1000 dilution in LICOR blocking buffer or 5% milk in TBST) LC3 (PM036; MBL International), Atg3 (ab108251; Abcam), Runx2 (ab236639; Abcam), AMPKα (D5A2; Cell Signaling Technology), Phospho-AMPKα (Thr172) (40H9; Cell Signaling Technology), osterix (AF7580; Affinity Biosciences), Osteocalcin (ab93876; Abcam), bone sialoprotein (DF7738; Affinity Biosciences). Blots were subsequently incubated in goat anti-rabbit IRDye 680RD (926-68071; Thermo Fisher Scientific) or HRP conjugated goat anti-rabbit IgG (P0449; Dako) for 1 h. Blots were then imaged using an Odyssey CLx Infrared Imaging System (Li-COR) or developed by the GeneGenome system (Syngene).
Membranes were then washed reprobed for β-actin expression (1:1000; 4970; Cell Signaling Technology). For Atg3 studies, β-actin expression was determined on a parallel membrane due to molecular weights.
Nitrocellulose membranes were probed overnight at 4°C with primary antibodies (1:1000 dilution in LICOR blocking buffer or 5% milk in TBST) LC3 (PM036; MBL International), Atg3 (ab108251; Abcam), Runx2 (ab236639; Abcam), AMPKα (D5A2; Cell Signaling Technology), Phospho-AMPKα (Thr172) (40H9; Cell Signaling Technology), osterix (AF7580; Affinity Biosciences), Osteocalcin (ab93876; Abcam), bone sialoprotein (DF7738; Affinity Biosciences). Blots were subsequently incubated in goat anti-rabbit IRDye 680RD (926-68071; Thermo Fisher Scientific) or HRP conjugated goat anti-rabbit IgG (P0449; Dako) for 1 h. Blots were then imaged using an Odyssey CLx Infrared Imaging System (Li-COR) or developed by the GeneGenome system (Syngene).
Membranes were then washed reprobed for β-actin expression (1:1000; 4970; Cell Signaling Technology). For Atg3 studies, β-actin expression was determined on a parallel membrane due to molecular weights.
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