The lysis buffer which contains 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100 and protease inhibitor was used to re-suspend the pretreated cells. The estimation of the protein content was accomplished by a BCA kit (Applygen). Each sample (60 μg of protein) was subjected to SDS-PAGE and then transferred onto nitrocellulose membrane. Blots were blocked in a blocking buffer containing 5% (wt/v) non-fat milk, 0.1% (v/v) Tween-20 in 10 mM TBS, incubated overnight with primary antibodies at 4 °C on a shaker, incubated with an appropriate secondary antibody for 1 h at room temperature on a shaker and subsequently scanned on a Typhoon Trio Variable Mode Imager. Pretreated cells were re-suspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100 and protease inhibitor. The protein content was estimated using a BCA kit (Applygen). Each sample (60 μg of protein) was subjected to SDS-PAGE and then transferred onto nitrocellulose membrane. Blots were blocked in a blocking buffer containing 5% (wt/v) non-fat milk, 0.1% (v/v) Tween-20 in 10 mM TBS, incubated with primary antibodies overnight at 4 °C, incubated with an appropriate secondary antibody for 1 h at room temperature and subsequently scanned on a Typhoon Trio Variable Mode Imager. The uncropped and unprocessed scans are shown in Supplementary Figs. 18 and 33.
Bca kit
Manufactured by Applygen
Sourced in China
The BCA kit is a laboratory reagent used for the colorimetric detection and quantification of total protein content in a sample. It functions by utilizing the bicinchoninic acid (BCA) assay principle to measure the reduction of copper ions, which is proportional to the amount of protein present.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Applygen (2 mentions)
Anti actin, by Cell Signaling Technology (2 mentions)
Ab216327, by Abcam (1 mentions)
Ab96587, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Specific assay kit, by Nanjing Jiancheng (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Ab208035, by Abcam (1 mentions)
34 protocols using bca kit
1
Western Blot Analysis of Protein Expression
2
Colon Protein Expression Analysis
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ZO-1 and occludin in the colon were assessed by a Western blot. RIPA buffer (Thermal Scientific, USA) was used to homogenize the colon tissue. Then, the sample was centrifuged and prepared to measure the protein concentrations by BCA kits (P1511, Applygen Technologies Inc., Beijing, China). Each sample was loaded to the 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After being blocked for 4 h in a Tris-buffered saline/Tween 20, the membrane was incubated with primary antibodies of ZO-1 (1:1000; ab96587, Abcam), occludin (1:1000; ab216327, Abcam), and β-actin (1:5000; ab8227, Abcam) at 4 °C overnight. After that, the membrane was incubated with the secondary antibody. The immunoreactive protein bands were visualized using a Clarity Western ECL substrate kit (Sigma, CA, USA). Scion Image (Frederick, MD, USA) was used to perform the densitometry analysis of protein bands. The values were normalized against the intensity of β-actin (Figure 1 ).
3
Ferroptosis Pathway Regulation in BV2 and OPC
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Mouse BV2 and OPC cell lines were purchased from Guangzhou Cellcook Biotech Co., Ltd., Guangzhou International Biological Island Fer-1 (cat. no. HY-100579) and Nec-1 (cat. no. HY-15706) were purchased from MedChemExpress, Pudong New Area, Shanghai. ROS (cat. no. E004-1-1), malondialdehyde (cat. no. A003-1-2), and glutathione (cat. no. A006-2-1) were obtained from Nanjing Jiancheng Bioengineering Institute, Qinhuai, Nanjing. The Iron Assay Kit (cat. no. E1042) was supplied by Applygen, Changping, Beijing and BCA kits (cat. no. KGP903) and the CCK-8 Assay Kit (cat. no. KGA317) were provided by KeyGEN BioTECH, Jiangning, Nanjing, Jiangsu Province. DMEM (cat. no. 220511) and FBS (cat. no. 16140071) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, Massachusetts). TNF-α (cat. no. ADI-901-099) ELISA kits were acquired from Neobioscience TechnologyCo, Ltd., Nanshan, Shenzhen, RIPK1 (cat. no. 3493), p-RIPK1 (cat. no. 53286), RIPK3 (cat. no. 95702), p-RIPK3 (cat. no. 91702), MLKL (cat. no. 37705), p-MLKL (cat. no. 37333), GAPDH (cat. no. 5174) and TNF-α (cat. no. 11948) were provided by Cell Signaling Technology, Danvers, Massachusetts,USA. GPX4 (cat. no. ab125066) was supplied by Abcam, Shanghai, China.
4
Oxidative Stress Evaluation in Lens
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After experiments, the lenses were washed with cold 0.9% saline, dried with filter paper, and weighed. Then, the lenses (n = 6 per group) were homogenized in a glass homogenizer with 0.9% saline at a ratio of 1:9. The homogenates were centrifuged at 3000×g for 15 min at 4 °C, and the supernatants were collected for assays. GSH content and total SOD and CAT activities were measured using specific assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. The protein content of the supernatant was determined using a BCA kit (Applygen Technologies Inc., Beijing, China). For all assays, the activity was calculated as the fold change from the control.
5
Western Blot Analysis of Liver and Muscle Proteins
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The liver or skeletal muscle tissue was homogenized using RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor cocktail (Merck, Darmstadt, Germany), and then centrifuged for 15 min at 12,000× g (4 °C). The protein content of the supernatant was quantified with a BCA kit (Applygen, Beijing, China). The protein samples were subjected to SDS-PAGE, and thereafter transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 1% BSA TBS-T solution, the membranes were incubated with primary antibodies against GAPDH (Beyotime, AG019-1, 1:1000), SELENOT (Abcam, ab176192, 1:1000), Type I iodothyronine deiodinase (DIO1; Santa Cruz, Dallas, TX, USA, sc-515198, 1:100), Glutathione S-transferase A2 (Gsta2; Thermo Fisher Scientific, PA5-100255, 1:500) and Glycogen [starch] synthase, liver (Gys2; Santa Cruz, sc-390391, 1:100), respectively, and subsequently secondary antibodies conjugated with horseradish peroxidase (Biosharp, Hefei, Anhui, China, BL001A/BL003A). Finally, the membranes were visualized with ECL kit (Millipore, Billerica, MA, USA) using a Tanon 5200 Automatic Chemiluminescence Imaging Analysis System (Tanon, Shanghai, China).
6
Western Blot Analysis of Apoptosis Markers
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The total protein was isolated by RIPA buffer (Beyotime, Shanghai, China). The concentrations of proteins were detected via a BCA kit (Applygen Technologies Inc., China). Proteins were separated on a 10% SDS-PAGE gel, and transferred to a PVDF membrane. After blocking via 5% skimmed milk powder, the membranes were probed with primary antibodies, including anti-YOD1, CDK4, CyclinD1, Bax, Bcl-2, Caspase- 3 (1: 1000; Cell Signaling Tech., Beverly, MA, United States), and anti-β-actin (1: 5000, Cell Signaling Tech.), at 4°C overnight. Then, secondary antibodies (1: 5000, Cell Signaling Tech.) were used to incubate the membranes at 20°C for 2 h. Protein bands were visualized by the enhanced chemiluminescence (Millipore, Billerica, MA, United States).
7
Immunoblotting Protein Expression Analysis
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Cells were lysed in RIPA lysis buffer (Applygen, Beijing, China) containing protease inhibitors (Applygen, Beijing, China) and phosphatase inhibitors (Applygen, Beijing, China). Protein concentrations were measured using a BCA kit (Applygen, Beijing, China). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 45 min at a constant voltage of 80 V and 45 min at 120 V, the separated proteins were transferred to PVDF membranes (Millipore, Sigma, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk powder at room temperature for 1.5 h and then incubated overnight at 4 °C with primary antibodies: rabbit anti-ADAM9 mAb (1:1000, 4151, Cell Signaling Technology, Danvers, MA, USA), mouse-anti-IL-17A mAb (1:1000, 66148-1-lg, Proteintech, Rosemont, IL, USA), rabbit anti-JNK mAb (ab208035, Abcam, Cambridge, MA, USA), and rabbit anti-phospho-JNK mAb (ab76572, Abcam, Cambridge, MA, USA). HRP-labeled goat anti-rabbit or goat anti-mouse lgG (ZSGB-Bio, Beijing, China) was used as secondary antibodies. Immunoreactive bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Sigma, Billerica, MA, USA) and analyzed using ImageJ software (National Institutes of Health, MD, USA).
8
Western Blot Analysis of Apoptosis Markers
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Cells were harvested and lysed in RIPA containing proteinase and phosphatase inhibitors. The concentration of proteins in cell lysates were quantified by BCA kit (Applygen Technologies, China) following the instruction. Equal amounts of cell lysates (20 µg protein/lane) were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Membranes were blocked and then probed with the appropriate primary antibody [PYCR1, 1:5,000 (Proteintech); PARP, 1:1,000 (Proteintech); Caspase-3, 1:1,000 (CST); cleaved-Caspase 3, 1:1000 (CST); p-PI3K, 1:2,000 (Proteintech); p-Akt, 1:4,000 (Proteintech); Akt, 1:1,000 (Proteintech); β-actin, 1:8,000 (Applygen)] in blocking buffer overnight at 4 °C. The bound antibodies were detected with IRDye 800CW-conjugated goat anti-rabbit IgG or anti-mouse IgG (at 1:10,000 dilution; LI-COR Bioscience, Lincoln, NE, USA) and visualized with Odyssey 290 infrared imaging system (LI-COR Bioscience).
9
Inflammatory Mediators in Bladder Tissue
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Bladder specimens were isolated from the rats and then lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Supernatants were then harvested followed by centrifugation at 12,000 g. Protein concentration of supernatants was measured by BCA kit (Applygen, Beijing, China). Levels of Tumor Necrosis Factor Alpha (TNF-α), interleukin (IL)-6, and IL-1β were determined using ELISA kits (ExCell Biology, Inc., Shanghai, China). Serum level of Prostaglandin E2 (PGE2) was also determined using ELISA kit (ExCell Biology, Inc.).
10
Corneal Fibroblast Mechanotransduction by IL-1β
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Corneal fibroblasts were seeded on six-well Bioflex® plates (Flexcell Int. Corp., Hillsborough, NC, USA) with flexible bottoms coated with type I collagen. When the cell confluency reached 70 %, the medium was removed and replaced with DMEM/F12 medium containing 1 % FBS and recombinant human IL-1β (0, 0.2, 0.4 and 0.8 ng/mL). Then, the cells were subjected to a mechanical strain of downward deformation by a computer-controlled vacuum using a Flexcell® Tension Plus™ FX-4000™ system (Flexcell Int. Corp., Hillsborough, NC, USA) with an elongation rate of 5, 10 or 15 % that was alternatively applied (0.1 Hz, 36 h, sine waveform).
The cell culture supernatants were collected at 36 h as protein samples for zymography or western blot analysis. The protein concentration was measured using BCA kit (Applygen Technologies Inc., Beijing, China).
The cell culture supernatants were collected at 36 h as protein samples for zymography or western blot analysis. The protein concentration was measured using BCA kit (Applygen Technologies Inc., Beijing, China).
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