Cells were lysed and sonicated in the buffer containing 1% Triton X-100, 10 mmol/L TrisHCl (pH 7.4) and proteases/phosphates inhibitors (Roche Diagnostics, USA), electrophoresed in 10% SDS-PAGE gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (PVDF) probed with primary antibodies to KPNA2. Subsequently, the membrane was probed with horseradish–peroxidase conjugated secondary antibodies. Then the blots were detected and visualized by a chemiluminescence detection system (Pierce, Rockford, IL, USA). The primary antibodies were used: anti-KPNA2 (Abcam, Cambridge, MA, USA), anti-c-myc(Abcam, Cambridge, MA, USA), anti-histoneH3 (Boster, Pleasanton, CA, USA); anti-E2F1 (Abcam, Cambridge, MA, USA), and anti-GAPDH (Boster, Pleasanton, CA, USA).
Anti gapdh
Manufactured by Boster Bio
Sourced in China, United States
Anti-GAPDH is a primary antibody designed to detect the presence of the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein in biological samples. GAPDH is a widely used internal control and housekeeping gene, making Anti-GAPDH a valuable tool for research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (8 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Anti β actin, by Boster Bio (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Bca assay kit, by Beyotime (3 mentions)
Anti β catenin, by Boster Bio (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Anti cyp2e1, by Boster Bio (2 mentions)
Anti nrf2, by Bioss Antibodies (2 mentions)
Anti lamin b, by Bioss Antibodies (2 mentions)
Anti kpna2, by Abcam (1 mentions)
Anti e2f1, by Abcam (1 mentions)
Anti c myc, by Abcam (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti n cadherin, by Proteintech (1 mentions)
Anti bak, by ABclonal (1 mentions)
Anti bcl 2, by Huabio (1 mentions)
Anti c myc, by Affinity Biosciences (1 mentions)
Anti survivin, by Novus Biologicals (1 mentions)
36 protocols using anti gapdh
1
Western Blot Analysis of KPNA2 Expression
2
Comprehensive Protein Analysis Techniques
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SDS‐PAGE and western blot were conducted using the following antibodies: anti‐GAPDH (BM3874, Boster), anti‐TRIM55 (A15917, Abclonal), anti‐MMP2 (orb12416, Biorbyt), anti‐MMP9 (27306‐1‐AP, Proteintech), anti‐N‐Cadherin (22018‐1‐AP, Proteintech), anti‐Bak (A0498, Abclonal), anti‐Bax (sc‐7480), anti‐Bcl2 (ET7110‐51, HuaBio), anti‐Caspase 3 (ET1602‐39, HuaBio), anti‐c‐Myc (AF0358, Affinity), anti‐p21/Cip1 (27296‐1‐AP, Proteintech), anti‐E‐Cadherin (20874‐1‐AP, Proteintech), anti‐P27/Kip1 (NBP1‐32213, Novus), anti‐survivin (NBP2‐48494, Novus), mouse anti‐HA‐Tag mAb (AE065, Abclonal), and mouse anti His‐Tag mAb (AE003, Abclonal). Protein bands were developed with a chemiluminescence kit (ThermoFisher).
3
NLRP3 Inflammasome Activation Assay
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Cells were collected and lysed in M-PER Mammalian Protein Extraction Reagent. All samples were normalized by their protein concentrations, separated in 10% SDS-PAGE gels, and then transferred to membranes (Washington, NY) using a wet transfer blotting system (Hercules, CA). The antibodies used for Western blotting were: anti-NLRP3 (Abcam), anti-caspase-1 (Abcam), and anti-GAPDH (Boster, Wuhan, China), at a dilution of 1:500. Protein bands were developed using ECL reagents, whose images were acquired using the BIO-RAD Imaging system. Western blot examination was repeated three times.
4
Ginseng Compound G‐Rb3 Protects Against Cisplatin‐Induced Toxicity
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G‐Rb3 (purity ≥ 98.5%, HPLC method) was isolated and purified from the leaves of Panax quinquefolium (American ginseng). Cisplatin was purchased from Sigma Chemicals with purity more than 99%. Compound C, rapamycin (Ram) and acetylcysteine (NAC) also were purchased from MedChemExpress Biotech and stored at −80°C in darkness. The commercial assay kits for determining reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), blood urea nitrogen (BUN) and creatinine (CRE) were bought from Nanjing Jiancheng Biological Research Institute. Haematoxylin and eosin (H&E) dying kit and Hoechst 33258 staining kit were obtained from Beyotime Co, Ltd. The immunohistochemically assay kits together with SABC‐DyLight488 immunofluorescence staining kits were obtained from BOSTER Biological Technology Co, Ltd. The primary rabbit monoclonal antibodies including anti‐LC3, anti‐BNIP3, anti‐β‐actin, anti‐GAPDH, anti‐Atg3, anti‐Atg5, anti‐Atg7 and anti‐p62 were all provided by BOSTER Biological Technology Co, Ltd. The rabbit anti‐AMPK, rabbit anti‐mTOR, rabbit anti‐Bax, Bcl‐2, Bad, caspase 3 and caspase 9 were acquired from Cell Signaling Technology. TUNEL commercial kit was purchased from Roche Applied Science. All other reagents and chemicals, unless indicated, were obtained from Beijing Chemical Factory.
5
Western Blot Analysis of CDC73 Protein
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Total protein was isolated from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and the protein concentrations were measured using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 20 µg of protein were resolved with 10% SDS-PAGE gel, transferred to polyvinylidene difluoride membranes and blocked in 5% skimmed dry milk in Tris-buffered saline (pH 7.4), containing 0.05% Tween-20. Subsequently, the membrane was incubated with anti-CDC73 antibody (catalog no. PB0587; 1:1,000; Boster Biological Technology, Wuhan, China) overnight at 4°C and then with anti-GAPDH (catalog no. BM3876; 1:3,000; Boster Biological Technology) for 2 h at room temperature as a loading control. Signals were detected by secondary antibodies labeled with horseradish peroxidase (HRP) and were visualized using an enhanced chemiluminescent kit (Beyotime Institute of Biotechnology).
6
Protein Extraction and Western Blot Analysis
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Total protein was extracted from bone marrow tissues and cultured cells using the RIPA lysis buffer (Beyotime, China). Approximately 25 µg proteins were separated by 12% SDS-PAGE and then transferred into PVDF membranes. After blocking with 5% lipid-free milk solution, the membranes were incubated with primary antibodies anti-INPP4B (1:1000; #ab14768, Abcam) and anti-GAPDH (1:2000; Boster, Wuhan, China) overnight at 4°C. Subsequently, the membranes were exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. Finally, the protein signals of targets were detected by using the ECL Kit (Millipore), and relative protein levels were quantified by using ImageJ software.
7
Cell Lysate Analysis and Immunoblotting Techniques
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Cell or tissue lysates and immunoblot analysis were performed as described previously [11 (link), 12 (link)]. Densitometric analysis was conducted using ImageJ software. The primary antibodies used include: anti-STIP1 (1:200, Boster Biotechnology, Wuhan, China), anti-ALK2 (1:400, Boster Biotechnology, Wuhan, China), anti-SMAD1/5 (1:200, Boster Biotechnology, Wuhan, China), anti-CTSK (1:500, Boster Biotechnology, Wuhan, China), anti-ERK1/2 (1:500, Boster Biotechnology, Wuhan, China), anti-Hsp90 (1:500, Boster Biotechnology, Wuhan, China), anti-Ki67 (1:500, Boster Biotechnology, Wuhan, China) and anti-GAPDH (1:500, Boster Biotechnology, Wuhan, China).
8
RNA methylation dynamics and regulation
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Reagents used were as follows: Transfection was done with Oligofectamine (Invitrogen Life Technologies, 12252–011) as recommended by the manufacturer. The following antibodies were used: anti-m6A antibody (Synaptic Systems, 202003), METTL3 (Proteintech, 15073–1-AP), METTL14 (Sigma-Aldrich, HPA038002), anti-GAPDH (Bosterbio, 0411), anti-FLAG (Sigma-Aldrich, F3165), anti-SQSTM1 (Sigma-Aldrich, P0068), rabbit polyclonal anti-LC3B antibody (Sigma-Aldrich, L7543), TFEB (Bethyl Laboratories, A303-673A), cleaved CASP3/caspase-3 (Abcam, Ab2302), PCNA (Abcam, ab18197), GFP (Abcam, ab13970), HNRNPD (Abcam, ab61193), ELAVL1 (Abcam, ab54987), TFEB-phospho-Ser142 (MilliporeSigma, ABE1971), phospho-AMPK-Thr172 (Cell Signaling Technology, 2535S), phospho-RPS6KB/p70 S6 Kinase-Thr389 (Cell Signaling Technology, 9205), ALKBH5 (Abcam, ab69325), FTO (Abcam, ab92821), and TUBA (Abcam, ab6046).
9
Combinatorial Therapeutic Assessment with MK-2206 and Finasteride
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MK-2206 2HCl and finasteride (Fin) were purchased from Shanghai Selleck Chemicals Co., Ltd. Shanghai, China. Organic reagents such as ethanol and acetonitrile were obtained from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China. Cell Cycle Assay Kit was acquired from Dojindo, Kumamoto, Japan. Primary antibodies included anti-β-Catenin, anti-GSK3β, anti-phospho-GSK3β, anti-GAPDH, and anti-β-tubulin (Boster Biological Technology, Pleasanton, CA, United States), anti-Bcl2, anti-Bax, and anti-Cyclin D1 (Abcam plc, Cambridge, United Kingdom), anti-Akt and anti-phospho-Akt (Cell Signaling Technology, Danvers, MA, United Ststes), anti-Wnt10b, anti-CDK2, anti-CDK4, and anti-P21 (ABclonal, Wuhan, China), and anti-ki67 (Proteintech, Wuhan, China). The chemiluminescence kit was obtained from Vazyme Biotech, Nanjing, China. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection kit was acquired from Promega Corporation, Madison, WI, United States.
10
Protein Expression Analysis of rBMSCs
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The rBMSCs were cultured in different culture medium for 3 days. For total protein extraction, rBMSCs were lysed in ice-cold RIPA buffer and centrifugated at 12 000 rpm for 10 min at 4 °C.28 (link) The amount of proteins was quantified using a BCA protein assay kit (Beyotime, China). By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Epizyme, China), the protein samples (10 μg) were separated and transferred to 0.22 μm pore-sized poly-vinylidene difluoride membranes (PVDF, Sangon, China). After being blocked with 0.1% tween containing 5% nonfat dry milk solution for 1 h at room temperature, the membranes were incubated overnight at 4 °C with anti-p-ERK1/2, ERK1/2 (1 : 1000, Abcam, UK), anti-RUNX-2 (1 : 1000, CST, USA), anti-GAPDH (1 : 1000, Boster, China) in the blocking buffer. After being incubated with the secondary antibodies for 1 h, specific protein bands were detected using an enhanced chemiluminescence detection system (Millipore, USA). For western blot analysis, GAPDH protein expression was used as an endogenous control for normalization.29 (link)
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