Liver samples were collected from six fish from each treatment. Western blotting was conducted using RIPA buffer (NCM Biotech, China) according to the method described previously (Li et al. 2020b (link)). The anti-CPT1a (1:800, 15184-1-AP, Proteintech), anti-LXRα/β (1:1000, sc-271064, Santa Cruz Biotechnology), anti-SREBP1c (1:1000, 66875-1-Ig, Proteintech), anti-LC3 (1:800, #4107, CST), anti-LAL (1:500, Custom Antibody Services from HuaAn Biotech, China), anti-IRβ (1:800, 20433-1-AP, Proteintech), anti-PI3K (1:800, CY6915, Abways), anti-p-PI3K (Tyr458/199) (1:800, #4228, CST), anti-AKT (1:800, #4091S, CST), anti-p-AKT (1:800, #4260S, CST), anti-mTORC1 (1:1000, #2972S, CST), anti-p-mTORC1 (1:1000, #2971S, CST), anti-S6 (1:1000, #2217S, CST), anti-p-S6 (1:800, AY0578, Abways), anti-GAPDH (1:3000, AB0036, Abways) and anti-α-Tubulin (1:1000, M1501-1, HuaAn Biotech) were used to measure the expressions of targeted proteins. The Western blotting images were obtained using an Odyssey CLx Imager (Licor, USA).
Ab0036
Manufactured by Abways
Sourced in China
The AB0036 is a precision laboratory balance designed for accurate weight measurements. It features a high-resolution digital display and a durable construction for reliable performance in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Zb 2301, by ZSGB-BIO (3 mentions)
D220014, by Sangon (3 mentions)
Whole protein extraction kit, by Keygen Biotech (2 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Sc 271064, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit 23225, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Keygen Biotech (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Af0120, by Affinity Biosciences (1 mentions)
Af6139, by Affinity Biosciences (1 mentions)
Ab129068, by Abcam (1 mentions)
Ab154856, by Abcam (1 mentions)
Ab56788, by Abcam (1 mentions)
Ab154244, by Abcam (1 mentions)
Ab11168, by Abcam (1 mentions)
Bsm 52396r, by Bioss Antibodies (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Superlumia ecl plus hrp substrate kit, by Abbkine (1 mentions)
10 protocols using ab0036
1
Western Blotting Analysis of Liver Proteins
2
Protein Extraction and Western Blot Analysis
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Cells were washed with sterilized PBS, lysed with 200 μl RIPA (PC101, EpiZyme Biotechnology) for 40 min, and centrifuged at 13,000 × g for 5 min, and the supernatant was collected. Protein concentrations were quantified by a bicinchoninic acid (BCA) protein assay (BCA Protein Assay Kit 23,225, ThermoScientific). After determining the protein concentration, 50 μl of 5× loading buffer was added and placed in boiling water for 10 min. A 25 μg protein sample was subjected to SDS‒PAGE electrophoresis, after which the protein was transferred to a 0.22 μm PVDF membrane and blocked with skim milk powder for 2 h, and the primary antibody was incubated at 4°C for 6 h. After the primary antibody incubation, the secondary antibody was incubated for 45 min after washing five times with TBST. After incubation, the cells were washed three times with TBST and exposed to the following antibodies: anti-Flag (14793, CST), anti-CALCOCO2 (ab124372, Abcam), anti-GFP (AB0047, Abways Technology), anti-GAPDH (AB0036, Abways Technology), anti-GST (2625, CST), anti-HA (3724, CST), anti-Flag (14793, CST), anti-IRF3 (4302, CST) and anti-β-actin (P68133, Abways Technology).
3
Protein Expression Analysis in bMECs
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The total protein of the bMECs was extracted with a whole protein extraction kit (KeyGEN, Nanjing, China), and its concentration was determined using a bicinchoninic acid protein assay kit according to the manufacturer’s instructions (KeyGEN, Jiangsu, China). A total of 100 μg protein extracts were loaded, then separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The extracts were blocked in a blocking solution at room temperature for 15 minutes, and the membranes were incubated with antibodies. Primary antibodies against GAPDH (AB0036, 1:3,000; Abways, Shanghai, China), proliferating cell nuclear antigen (PCNA) (D220014, 1:500; Sangon Biotech, China), and cyclin-dependent kinase2 (CDK2) (1:500; Sangon Biotech, China) were used, and the secondary antibody was goat anti-rabbit IgG (ZB-2301, 1:20,000; ZSGB-Bio, Beijing, China). A chemiluminescent ECL Western blot system (Tanon-5200; Tanon, Shanghai, China) was used for signal detection, and protein abundance was measured using Image J software.
4
Bax and Bcl-2 Expression in Myocardial Tissue
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According to the standard scheme, the expression of Bax and Bcl-2 in myocardial tissue was detected by Western blotting. Protein was extracted from myocardial tissue in a buffer containing RIPA lysate and protease inhibitor. The concentration of the protein was tested by BCA kit. The same amount of protein (30 μg) was transferred from SDS-polyacrylamide gel to polyvinylidene fluoride membrane. PVDF membrane was sealed for 1 h with 5% skim milk powder and incubated with primary antibody anti-Bax (AF0120, 1:2,000 dilution, Affinity, USA), anti-Bcl-2 (AF6139, 1:1,000 dilution, Affinity, United States), or anti-GAPDH (AB0036, 1:5,000 dilution, Abways, Shanghai, China) overnight at 4°C. PVDF membrane was washed 3 times with TBST for 10 min each time and incubated the second antibody labeled with HRP and shaked the bed for 1 h. The protein signal were detected by using enhanced chemiluminescence reagents (ECL) in Chemilluminescence Imaging System (ChemiScope 6,000 system pro, clinx, Shanghai, China) and analyzed by Quantity One software (Bio-Rad, Richmond, California, CA, United States).
5
Citronellal's Antioxidant and Mitochondrial Effects
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Citronellal (CT, Beijing Yuke Biotechnology Co., Ltd) was purchased form Beijing, China. streptozotocin (STZ), metformin (DMBG), acetylcholine (Ach) and sodium nitroprusside (SNP) were purchased from Signal-Aldrich, St. Louis, MO, USA. The kits for MDA (A003-1-2), NO (C009-2-1), ROS (E004-1-1), and SOD (A001-3-2) were obtained from Nanjing Jiancheng, Nanjing, China. Dihydroethidium (DHE, S0063, Beyotime), ROS assay kit (ROS, S0033S, Beyotime), and JC-1-mitochondrial membrane potential assay kit (C2003S, Beyotime) were obtained from Beyotime, shanghai, China. The anti-NOX2 antibody (ab129068, Abcam), anti-NOX4 antibody (ab154244, Abcam), anti-DRP1 antibody (ab56788, Abcam), anti-TRPM2 antibody (ab11168, Abcam) and anti-VDAC (ab154856, Abcam) were obtained from Abcam, Cambridge, MA, USA. The anti-NHE1 antibody (bs-0505R, Bioss) and anti-α-SMA antibody (bsm-52396R, Bioss) were obtained from Bioss, Beijing, China. The anti-GAPDH antibody (AB0036, Abways) was acquired from Shanghai, China.
6
Quantification of Protein Expression in BMECs
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Total protein was extracted from BMECs using a whole-protein extraction kit (KeyGEN, Nanjing, China), and protein concentration was determined using a BCA protein assay kit (KeyGEN, Nanjing, China) according to manufacturer’s instructions. A total of 100 μg protein was loaded onto 10% SDS polyacrylamide gels, separated by electrophoresis, and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in a protein free rapid blocking buffer (Epizyme Biotech, Shanghai, China) at room temperature for 15 min and the membranes were then incubated with antibodies targeting proteins of interest. Primary antibodies were used to detect GAPDH (AB0036, 1:3000, Abways), PTPRF (A5444, 1:2000, ABclonal), PCNA (D220014, 1:500, Sangon Biotech) and CDK2 (1:500, Sangon Biotech), and goat anti-rabbit IgG (ZB-2301, 1:20,000, ZSGB-Bio) was used as the secondary antibody. A chemiluminescent ECL Western blot system (Tanon-5200, China) was used for signal detection, and protein was quantified using Image J software. Three biological and technical replicates in parallel of each treatment were measured.
7
UCP3 Protein Expression Analysis
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The heart tissue was homogenized in RIPA lysis buffer (Cwbiotech) and supplemented with 1 mM PMSF (Sigma) and 1× protease/phosphatase Inhibitor Cocktail (Cwbiotech). After quantification, each sample was denatured in SDS–polyacrylamide gel electrophoresis (PAGE) Loading Buffer (Cwbiotech) and boiled for 5 min. A total of 50 μg of protein was loaded in 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore). After blocking in TBST solution with 5% non-fat milk for 2 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies: anti-UCP3 (Zen-bio, 516996, 1:1,000) and anti-GAPDH (Abways, AB0036, 1:5,000). Membranes were then washed and incubated with species-specific HRP-conjugated secondary antibodies (Abways, AB0101, 1:5,000) at 1:5,000 in TBST with 5% non-fat milk for 1 h at room temperature. The blot was visualized by adding Superlumia ECL Plus HRP substrate kit (Abbkine) and then captured and analyzed by UVP Bio Imaging Systems (Sagecreation).
8
Western Blot Analysis of BMEC Proteins
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The BMECs were collected in 0.25% trypsin (Solarbio, Beijing, China) and Western blot analysis was performed using standard techniques reported by Hou [24 (link)]. The protein concentration was determined using a BCA protein determination kit (KeyGEN BioTECH Bio, Jiangsu, China). A total of 100 μg protein extracts were loaded, then separated using 10% SDS polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Epizyme Bio, Shanghai, China). The extracts were blocked in a blocking solution at room temperature for 15 min and the membranes were incubated with antibodies. Primary antibodies against GAPDH (AB0036,1:3000, Abways, Shanghai, China), PCNA (D220014, 1:500, Sangon Biotech, Shanghai, China), BAX (CY5059, 1:500, Abways, Shanghai, China), AMPK (AP0569, 1:500, Bioworld, Shanghai, China), and p-AMPK (BS4010, 1:500, Bioworld, Shanghai, China) were used, and the secondary antibody was goat anti-rabbit IgG (ZB-2301, 1:20000, ZSGB-Bio, Beijing, China). A chemiluminescent ECL Western blot system (Tanon-5200, Shanghai, China) was used for signal detection, and protein abundance was measured using ImageJ software.
9
SKOV3 and PEO-1 Cell Line Protein Analysis
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10-cm dishes were used to seed and culture SKOV3 and PEO-1 cells. Cells were collected after 48 h of drug treatment. RIPA lysis buffer (Sigma) was used to extract proteins, and BCA assay was used to determine protein concentrations. SDS-PAGE (Sanguang Bioengineering Technology Services, Shanghai, China) was performed by means of the operating instructions on the Cell Signaling Technologies website. Western blotting was performed using antibodies against BCL2 (T40056F, ABWAYS), BCL-XL (2764S, Cell Signaling Technology), CyclinB1 (12231S, Cell Signaling Technology), CyclinE1 (4129 T, Cell Signaling Technology), c-MYC (CY5150, ABWAYS), γ-H2AX (9718S, Cell Signaling Technology), GAPDH (AB0036, ABWAYS). Anti-rabbit 800 and mouse 800 (LI-COR Biosciences). Imaging of the membrane was carried out using an Odyssey infrared imaging system (LI-COR Biosciences), and measurements were made using Image Studio Lite software.
10
Scaffold-Mediated Osteoblast Protein Analysis
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Cells were seeded on BC, CA, BC/CA scaffolds for 7 and 14 days. The culture medium was then removed and cells were washed with warm PBS three times followed by addition of 100 μL of mixing solution of RIPA and PMSF (Solarbio, China), and complete lysis of the cells for 10 min. The total protein was kept at 1 μg/μL. Then protein electrophoresis was performed in PAGE gel and transferred to PVDF membranes. The membranes were blocked with 3% skimmed milk and then hybridized using diverse primary antibodies. The rabbit-derived ALP anti-body (AB3242, Abways, China) and mouse-derived GAPDH (AB0036, Abways, China) incubated at 4 °C overnight. The next day, goat anti-rabbit and goat anti-mouse second antibody (Jackson, America) were incubated for 1 h at room temperature. The pictures were obtained with Tanon Imager (5200, China).
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