Cell samples were lysed using a lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration was determined using a BCA protein assay kit (Beyotime). The proteins were separated on 10% SDS PAGE gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with primary antibodies overnight at 4°. The primary antibodies used were rabbit anti-LC3B (L7543, 1:1000, Sigma-Aldrich), anti-SQSTM1/p62 (ab91526, 1:1000, Abcam, Cambridge, MA, USA), anti-cleaved PARP (#9546, 1:2000, Cell Signaling, Danvers, MA, USA), anti-HMGB1 (ab77302, 1:1000; Abcam), and anti-β-actin (ab189073, 1:1000; Abcam). After washing 3 times, the membranes were incubated in HRP-conjugated secondary antibodies for another 1 h at room temperature. Then, the protein signals were detected using the ECL Western blotting substrate (Promega, Madison, WI, USA).
Ab77302
Manufactured by Abcam
Sourced in United States
Ab77302 is a laboratory equipment product offered by Abcam. It serves as a core function, without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab54741, by Abcam (2 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Proteinase and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Ecl western blotting substrate, by Promega (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ab91526, by Abcam (1 mentions)
Ab189073, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
L7543, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Beyotime (1 mentions)
Sc 57534, by Santa Cruz Biotechnology (1 mentions)
Sc 59737, by Santa Cruz Biotechnology (1 mentions)
Ab128915, by Abcam (1 mentions)
Ab40754, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Kgp1050, by Keygen Biotech (1 mentions)
Ab18414, by Abcam (1 mentions)
Ab18447, by Abcam (1 mentions)
5 protocols using ab77302
1
Western Blot Analysis of Autophagy Markers
2
Western Blot Analysis of Protein Expression
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Total protein was extracted with the active protein extraction kit (KGP1050; Nanjing KeyGen Biotech, Nanjing, China), and protein levels were quantified with the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and transferred to polyvinylidene fluoride membranes (Beyotime Biotechnology). After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated overnight at 4 °C with primary antibodies against HMGB1 (ab77302; Abcam, Cambridge, MA, USA), p-β-catenin (sc-57534; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (sc-59737; Santa Cruz Biotechnology), cyclin D1 (ab40754; Abcam), and GAPDH (ab128915; Abcam), which were diluted to 1:1,000. The, the membranes were incubated with goat anti-rabbit (ab205718; Abcam) or goat anti-mouse (ab6789; Abcam) HRP-conjugated secondary antibodies at a dilution of 1:5,000 for detection. The bands were visualized using enhanced chemiluminescence solution (Pierce; Thermo Fisher Scientific, Inc.). GAPDH was used as a loading control.
3
Investigating 8-nitroG Formation Mechanisms
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To examine the role of HMGB1 and RAGE in 8-nitroG formation, A549 cells were pretreated with 10 μg/ml anti-HMGB1 (ab77302, Abcam Cambridge, UK) and 10 μg/ml anti-RAGE (ab54741, Abcam, Cambridge, UK) antibodies. We also used the corresponding isotype control IgGs [mouse IgG1 (ab18447, Abcam) for anti-HMGB1 antibody and IgG2a (ab18414, Abcam) for anti-RAGE antibody] to confirm the specificity of these antibodies. Then the cells were incubated with 200 ng/ml of In2O3, ITO and InCl3 for 4 h. Then 8-nitroG formation was examined by immunocytochemistry as described above.
4
HMGB1 and RAGE Protein Detection
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Total proteins were extracted from the cells via treatment with lysis buffer and benzylsulfonyl fluoride (PMSF) (both from Beyotime, Shanghai, China), and then the protein supernatants were centrifuged at 10000 rpm for 10 min (at 4°C) to remove tissue fragments from sediments. The precipitate was then quantified using a bicinchoninic acid protein assay. Next, protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membrane was blocked with 5% fat-free milk for 2 hours, and incubated with either anti-HMGB1 antibody (ab77302, 1:1000 dilution, Abcam, Cambridge, MA, USA) or anti-RAGE antibody (ab54741, 1:200 dilution, Abcam) at 4°C, overnight. The blots were incubated with horseradish peroxidase-conjugated secondary antibodies (ab205718, 1:2000 dilution, Abcam) at room temperature (25°C) for 1 hour with continuous shaking. After washing, proteins within Western blots were detected using an enhanced chemiluminescence kit (Millipore, MA, USA), and then the blots were exposed to an X-ray film. Bands were quantified using Fluorchem (9900) (Alpha Innotech, Sam Leandro, CA, USA) equipment, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
5
Western Blot Analysis of Articular Cartilage Proteins
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Total proteins of cells and mouse articular cartilage tissues were isolated with RIPA buffer containing 1% protease inhibitors (Sigma-Aldrich). Equal amount of each protein sample was loaded on 12.5% SDS-PAGE and then transferred to PVDF membranes (Millipore). Blocking was done by incubating the membranes with TBST containing 5% slim milk at 37 °C for 2 h and the corresponding primary antibodies at 4 °C for overnight. Membranes were then incubated with HRP-conjugated secondary antibodies (1:2000, Abcam) at RT for 1 h. Proteins were detected with an enhanced chemiluminescence (ECL) kit (Pierce). Primary antibodies used were anti-β-actin (1:1000, Abcam, ab8227), anti-p65 (1:1000, Abcam, ab16502), anti-IkBα (1:1000, Abcam, ab76429), and anti-HMGB1 (1:1000, Abcam, ab77302). Proteins were quantitatively analyzed and normalized relative to β-actin using Image-Pro Plus software (v6.0) (Media Cybernetics).
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