The soluble protein extracts were separated by SDS-PAGE on a 10%–20% gradient gel and transferred to a Polyvinylidene difluoride (PVDF) membrane. Each blot membrane was incubated as per instruction with primary antibodies. The primary antibodies against Bcl2 (ab692), Bax (ab7977), Caspase 3 (ab32351), Caspase 9 (ab32539), and β-actin (ab63982) were purchased from Abcam. The blots were incubated with secondary goat anti-rabbit antibody (Abcam, ab63982) for 1 h at room temperature. Protein bands were detected on X-ray film using an enhanced chemiluminescence detection system.
Ab32539
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab32539 is a mouse monoclonal antibody that recognizes the GAPDH protein. This antibody is suitable for use in various laboratory applications, including Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (24 mentions)
Ab13847, by Abcam (17 mentions)
Ab2302, by Abcam (11 mentions)
Ab2324, by Abcam (11 mentions)
Ab32124, by Abcam (11 mentions)
Ab32351, by Abcam (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ab8245, by Abcam (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ripa buffer, by Beyotime (5 mentions)
Ab59348, by Abcam (5 mentions)
Ab692, by Abcam (4 mentions)
Ab181602, by Abcam (4 mentions)
Ab8227, by Abcam (4 mentions)
Ab32042, by Abcam (4 mentions)
Ab109268, by Abcam (3 mentions)
Ab182858, by Abcam (3 mentions)
Ab25901, by Abcam (3 mentions)
Ab17942, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
53 protocols using ab32539
1
Western Blot Analysis of Apoptosis Markers
2
Apoptosis and Wnt5a Pathway Evaluation
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PPI (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; batch number, 111590-200402), cis-platinum (DDP; Yunnan, China), reverse transcription-polymerase chain reaction (RT-PCR) kit (PrimeScript™ RT reagent kit; Takara Biotechnology Co., Ltd., Dalian, China), primers (synthesized by Takara Biotechnology Co., Ltd.) and PCR kit (SYBR Premix Ex Taq™II: catalog no., DRR820A; Dalian, China) were used in the present study, as well as primary antibodies against Caspase-9 (ab32539), C-jun (ab31419) and Wnt5a (ab72583), which were obtained from Abcam (Cambridge, UK) and used at 1:80 dilution. The secondary antibody, which was the working solution from the AuraStain SP Mouse/Rabbit IHC test kit (P003IH-1) was acquired from Auragene Bioscience Corporation, Inc. (Changsha, China).
3
Immunodetection of Orexin and Related Signaling
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Mouse monoclonal anti-orexin-A antibody (sc-80263) and goat polyclonal anti-orexin receptor-1 antibody (sc-8072; 1:2,000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States); rabbit polyclonal anti-prepro-Orexin Antibody (ab3096; 1:2,000), rabbit monoclonal anti-phospho-mTOR antibody(ab109268; 1:2,000), and rabbit monoclonal anti-caspase-9 antibody(ab32539; 1:2,000) were from Abcam company (Cambridge, United Kingdom); mouse monoclonal p-Akt antibody (66444-1; 1:2,000), mouse monoclonal Akt antibody (60203-2; 1:2,000), rabbit polyclonal anti-mTOR antibody (20657-1; 1:2,000), rabbit polyclonal anti-Bcl-2 antibody (12789-1; 1:2000), and rabbit polyclonal anti-c-Myc antibody (10828-1; 1:2,000) were purchased from ProteinTech Group Inc. (Rosemone, IL, United States); anti-GAPDH antibody (100242; 1:5,000) was purchased from Sino Biological Inc. (Beijing, China). The secondary antibodies were from Jackson ImmunoResearch Laboratories Inc. (1:5000; West Grove, PA, United States). Orexin A was obtained Sigma-Aldrich (St. Louis, MO, United States). The OX1R antagonist, SB408124 was from Tocris Bioscience (Bristol, United Kingdom); the inhibitor of the Akt pathway, MK-2206, was obtained from Selleck Company (Houston, TX, United States).
4
Mitochondrial Dynamics and Apoptosis Regulation in Liver Cells
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The liver tissue and HepG2 cells were lysed by RIPA lysis solution (Beyotime, China) to extract the protein. The protein concentration was detected by BCA reaction kit. After quantitative analysis, the total protein was denatured in this study. SDS-Page gel was used for electrophoresis, electrophoresis apparatus (Bio-RAD, USA) was adjusted to 120 V for electrophoresis, PVDF membrane (Millipore, USA) was used for membrane transfer, and skim milk (Sigma, USA) for blocking. Primary antibodies (Abcam, UK): Sirt1 (1:1000; ab189494), optic atrophy 1 (Opa1, 1:1000; ab157457), mitofusin 2 (Mfn2, 1:1000; ab124773), Drp1 (1:1000; ab184247), NRF1 (1:1000; ab34682), mitochondrial transcription factor A (TFAM, 1:1000; ab252432; Abcam; UK), Bcl-2 (1:2000; ab182858), cleaved-caspase 3 (1:500; ab2302), BCL2-associated X protein (Bax, 1:1000; ab32503), cleaved-caspase 9 (1:2000; ab32539) and GAPDH (1:2500; ab9485) were then added overnight to incubate. The next day, goat anti-rabbit antibody (1:2000; ab288151) was incubated for 1 h with slow shaking at 25 °C. The immunoreactive bands were visualized by intensive chemiluminescent reagent. The gray value was analyzed by ImageJ software.
5
Apoptosis Signaling Pathway Proteins Analysis
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Cells were lysed by using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor to obtain total proteins. Protein concentrations were assessed under a Bio-Rad DC Protein Assay Kit (Yuwei Biotechnology, Guangzhou, China). After that, proteins were separated by SDS/PAGE and then moved on to the PVDF membrane. The membrane was sealed tightly with fat-free milk, followed by co-cultured with specific primary antibodies: anti-GPX1 (ab22604, Abcam, Cambridge, U.S.A.), anti-total caspase-3 (ab13847, Abcam), anti-cleaved caspase-3 (ab2302, Abcam), anti-total caspase-9 (ab32539, Abcam), anti-cleaved caspase-9 (ab2324, Abcam) and anti-GAPDH (ab8245, Abcam). After being incubated with secondary antibodies, the membrane was washed, and the protein bands were observed via chemiluminescence detection system and quantified via ImageJ software.
6
Western Blot Protein Analysis Protocol
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All proteins were extracted by RIPA buffer (Solarbio Life Science, Beijing, China) mixed with protease inhibitors on ice plates. Then, protein concentration was qualified by a BCA protein assay kit purchased from Solarbio Life Science. Next, equal amounts of protein were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% BSA (Sigma-Aldrich) and incubated with primary antibodies at 4°C overnight. On the second day, the membranes were washed with 1x TBS solution and incubated with the secondary antibody conjugated with HRP at normal temperature for 1 hour. Finally, the results were determined using the ChemiDocTM Imaging System (Bio-Rad, Hercules, CA, USA). The following primary antibodies were used: FOXP2 (20529-1-AP, 1:1000, ProteinTech), PCNA (ab29, 1:1000, Abcam), cyclin D1 (ab40754, 1:5000, Abcam), caspase-1 (sc-392736, 1:1000, Santa Cruz), caspase-3 (sc-7272, 1:1000, Santa Cruz), caspase-9 (ab32539, 1:1000, Abcam), GSDMD (ab209845, 1:1000, Abcam), GAPDH (1:1000, TransGen Biotech, Beijing), Flag-tag (D6W5B, CST), HA-tag (C29F4, CST), and β-actin (1:1000, TransGen Biotech, Beijing). The secondary protein was purchased from TransGen Biotech (Beijing).
7
Western Blot Analysis of Apoptosis Markers
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Total protein from HepG2 was obtained via using RIPA cell lysis buffer. Protein concentration was determined by a Nano drop spectrophotometer, and 20 μg protein samples were separated by 10 % SDS-PAGE, transferred to the PVDF membranes, and washed with TBS for 5 min. Following the addition of blocking buffer, anti-caspase 9 (1:2500, ab32539, abcam), anti-caspase 3 (1:1000, k009567p, solar bioscience) and anti-β-actin (1:2500, k200058m, solar bioscience) primary antibodies were added, and membranes were incubated overnight at 4 °C. The membranes were then incubated with the secondary antibody (1:5000, anti-rabbit IgG, 31460) for 2 h followed by HRP colorimetric substrate for 30 min at room temperature. Digital image software (iB-right™ CL1500 Imaging System, Invitrogen) was used for band quantification.
8
Western Blot Analysis of Osteoblast Markers
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The total proteins of MC3T3-E1 cells or BMSCs were extracted and lysed with RIPA buffer (Beyotime, China), and protein concentration was detected using a BCA protein assay kit (Solarbio, China). Then, a 20 μg total protein sample was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred to a PVDF membrane. After being blocked with 5% skimmed milk, the membrane was incubated at 37°C with the primary antibodies against caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), SIRT1 (ab110304, Abcam), BMP-2 (ab214821, Abcam), RANKL (ab45039, Abcam), OPG (ab73400, Abcam), IκBα (ab32518, Abcam), p-IκBα (AF2002, Affinity), p65 (ab207297, Abcam), p-p65 (AF2006, Affinity), IKKα (ab32041, Abcam), p-IKKα (AF3013, Affinity), NFATc1 (ab2796, Abcam), and cathepsin K (ab19027, Abcam) at 4°C overnight. After that, the membrane was further incubated with the secondary antibody at room temperature for 1 h. Then, the enhanced chemiluminescence (ECL) reagent (Solarbio, China) was used to visualize the protein bands, and the relative band density was semiquantified with the ImageJ software.
9
Western Blot Analysis of Apoptosis Markers
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EOCs were disposed by RIPA lysis buffer (Santa Cruz, Dallas, USA). The liquid was transferred into sterile EP tubes and placed on ice for 20 min. Afterwards, tubes were centrifuged at 12 000 rpm for 10 min. Protein concentrations were measured and compared with BCA. A mixture of equal parts of protein and sample buffer was subjected to SDS-PAGE. The extracted protein was transferred onto PVDF membranes. The PVDF membrane was blocked in blocking buffer (TBST, 5% fat-free milk) for 1 h at room temperature. The following primary antibodies were used for immunoblotting: p-JNK (Cell Signaling, 1: 1000, no.4668), JNK (Cell Signaling, 1: 1000, no.4672), Bcl-2 (Abcam, 1: 2000, ab32124), Bax (Abcam, 1: 2000, ab32503), cleaved caspase 3 (Abcam, 1: 2000, ab13585), and cleaved caspase 9 (Abcam, 1: 2000, ab32539). GAPDH was used as the internal reference. The PVDF membrane was incubated in the diluted primary antibodies at 4°C overnight and washed with TBST 3 times for 10 min. Then, the PVDF membrane was diluted in secondary antibody (Santa Cruz, Dallas, TX, USA, sc-33732, 1: 1000) at 4°C overnight. The PVDF membrane was demonstrated by Image Pro-Plus system (Media Cybernetics, Silver Spring, MD, USA).
10
Western Blot Analysis of Protein Markers
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Total proteins were extracted through a Total Protein Extraction Kit (KeyGen Biotech, Nanjing, China). Concentrations of protein were examined under BCA commercial kit (KeyGen). Proteins were separated using SDS-PAGE and then moved onto PVDF membranes (Millipore, Carlsbad, USA). Membranes were sealed in non-fat milk and then incubated with given primary antibodies: anti-MMP2 (1:1000, ab37150, Abcam, Cambridge, USA), anti-MMP7 (1:1000, ab5706, Abcam), anti-Bcl-2 (1:1000, ab32124, Abcam), anti-bax (1:1000, ab32503, Abcam), anti-caspase 3 (1:1000, ab13847, Abcam), anti-cleaved caspase-3 (1:1000, ab2302, Abcam), anti-caspase 8 (1:1000, ab25901, Abcam), anti-cleaved caspase-8 (1:1000, ab25901, Abcam), anti-caspase 9 (1:1000, ab32539, Abcam), anti-cleaved caspase-9 (1:1000, ab2324, Abcam), anti-E-cadherin (1:1000, ab15148, Abcam), anti-N-cadherin (1:1000, ab76057, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam). GAPDH was used as a measurement control for other proteins. Moreover, the membranes were co-cultured with goat anti-mouse IgG H&L (Cy3®) preadsorbed (1:2000, ab97035, Abcam) secondary antibodies for 1 h darkness. Chemiluminescence system (Invitrogen) was employed to observe the protein bands.
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