In vitro, followed by EF pre-treatment for 2 h, the grouped primary microglia were stimulated with LPS for 1.5 h or 24 h. In vivo, the ischemic penumbra tissues from grouped mice mentioned above were extracted on the third day of MCAO. Total proteins were extracted by using a pre-prep extraction solution consisting of lysis buffer (Thermo Fisher Scientific, Rockford, IL, United States) and 1% protease inhibitor, and concentrations were analyzed by a BSA kit (Beyotime). The protein samples mixed with 5 × loading buffer solution were separated with 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk at 37°C for 1 h, and incubated with specific primary antibodies anti-iNOS (1:1000), anti-COX-2 (1:1000), anti-NF-kB (1:1000), anti-phospho-NF-kB (1:1000), anti-phospho-IκBα (1:1000), anti- IκBα (1:1000) and anti-GAPDH (1:5000) at 4°C overnight. The membranes were washed with 1 × TBST (3 times, 5–10 min each) and incubated with secondary antibodies. The protein bands on the membranes were visualized using a Gel-Pro system (Tanon Technologies, China). The integrated band densities were quantified via ImageJ software (ImageJ-win64, NIH, United States).
Bsa kit
Manufactured by Beyotime
Sourced in China
The BSA kit is a laboratory equipment used for the determination of protein concentration. It provides a standardized method for quantifying the amount of protein present in a sample using the Bradford assay principle.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ab128870, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Sartorius (1 mentions)
Ab30682, by Abcam (1 mentions)
Ab45174, by Abcam (1 mentions)
Ab40793, by Abcam (1 mentions)
Sc 13551, by Santa Cruz Biotechnology (1 mentions)
Af0016, by Affinity Biosciences (1 mentions)
Af6261, by Affinity Biosciences (1 mentions)
Ripa protein lysis buffer, by Beyotime (1 mentions)
Anti ago2, by Abcam (1 mentions)
Anti c caspase3, by Abcam (1 mentions)
Anti gapdh, by Sino Biological (1 mentions)
One step animal tissue active protein extraction kit, by Sangon (1 mentions)
Ecl kit, by Fdbio (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Sds page, by Fdbio (1 mentions)
Ripa buffer, by Sangon (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
8 protocols using bsa kit
1
Microglia Activation and Inflammatory Signaling
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In vitro, followed by EF pre-treatment for 2 h, the grouped primary microglia were stimulated with LPS for 1.5 h or 24 h. In vivo, the ischemic penumbra tissues from grouped mice mentioned above were extracted on the third day of MCAO. Total proteins were extracted by using a pre-prep extraction solution consisting of lysis buffer (Thermo Fisher Scientific, Rockford, IL, United States) and 1% protease inhibitor, and concentrations were analyzed by a BSA kit (Beyotime). The protein samples mixed with 5 × loading buffer solution were separated with 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk at 37°C for 1 h, and incubated with specific primary antibodies anti-iNOS (1:1000), anti-COX-2 (1:1000), anti-NF-kB (1:1000), anti-phospho-NF-kB (1:1000), anti-phospho-IκBα (1:1000), anti- IκBα (1:1000) and anti-GAPDH (1:5000) at 4°C overnight. The membranes were washed with 1 × TBST (3 times, 5–10 min each) and incubated with secondary antibodies. The protein bands on the membranes were visualized using a Gel-Pro system (Tanon Technologies, China). The integrated band densities were quantified via ImageJ software (ImageJ-win64, NIH, United States).
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In vitro, followed by EF pre-treatment for 2 h, the grouped primary microglia were stimulated with LPS for 1.5 h or 24 h. In vivo, the ischemic penumbra tissues from grouped mice mentioned above were extracted on the third day of MCAO. Total proteins were extracted by using a pre-prep extraction solution consisting of lysis buffer (Thermo Fisher Scientific, Rockford, IL, United States) and 1% protease inhibitor, and concentrations were analyzed by a BSA kit (Beyotime). The protein samples mixed with 5 × loading buffer solution were separated with 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk at 37°C for 1 h, and incubated with specific primary antibodies anti-iNOS (1:1000), anti-COX-2 (1:1000), anti-NF-kB (1:1000), anti-phospho-NF-kB (1:1000), anti-phospho-IκBα (1:1000), anti- IκBα (1:1000) and anti-GAPDH (1:5000) at 4°C overnight. The membranes were washed with 1 × TBST (3 times, 5–10 min each) and incubated with secondary antibodies. The protein bands on the membranes were visualized using a Gel-Pro system (Tanon Technologies, China). The integrated band densities were quantified via ImageJ software (ImageJ-win64, NIH, United States).
2
Protein Quantification in BALF
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The leaked protein in the BALF was measured by the BSA kit (Beyotime), followed by the kit protocols.
3
Western Blot Analysis of Lipid Metabolism Proteins
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Total protein was extracted using RIPA protein lysis buffer (Beyotime Biotech, Jiangsu, China) with PMSF and complete protease and phosphatase inhibitor cocktail. Protein concentrations were determined by a BSA kit (Beyotime Biotech, Jiangsu, China). Fifty micrograms of protein was separated by gel electrophoresis and transferred to methylcellulose membranes. The membranes were blocked in 5% nonfat dried skimmed milk for 2 hours at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-HIF-2α (1:1000; #59973; CST), anti-MED15 (1:1000; 11566-1-AP; Proteintech), anti-SREBP1 (1:200; sc-13551; Santa Cruz), anti-SREBP2 (1:500; ab30682; Abcam), anti-FASN (1:1000; ab128870; Abcam), anti-ACC1 (1:1000; ab45174; Abcam), anti-ACLY (1:1000; ab40793; Abcam), anti-SCD1 (1:1000; #2794; CST), anti-PLK1 (1:1000; #4513; CST), Phospho-AKT (Ser473) (1:1000; AF0016; Affinity), AKT (1:1000; AF6261; Affinity), and anti-GAPDH (1:1000, #2118; CST). The membranes were incubated with secondary antibodies for 2 hours after being washed with tris-buffered saline and Tween 20 and were visualized by an enhanced chemiluminescence kit (Biological Industries, Israel).
4
Protein Extraction and Western Blot Analysis
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Cells were lysed with RIPA buffer (Sangon), and proteins were quantified using a BSA Kit (Beyotime, Shanghai, China). Tumor proteins were extracted by a One-Step Animal Tissue Active Protein Extraction kit (Sangon). Protein (20 μg) was loaded and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fdbio Science, Hangzhou, China) at 80 V for 30 min and then 120 V for 60 min followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore) at 300 mA for 90 min. After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Siglec-15 (1 : 1000, Santa Cruz), anti-GAPDH (1 : 5000, Sino Biological), anti-AGO2 (1 : 20, Abcam), anti-C-caspase3 (1 : 1000, Abcam), anti-caspase3 (1 : 1000, Abcam), and anti-Bcl-2 (1 : 1000, Abcam). After incubation with HRP-conjugated antibodies (1 : 5000, Sino Biological) for 2 h at room temperature, an ECL kit (Fdbio Science) was used for visualization of protein bands.
5
Oxidative Stress Assessment Protocol
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Oxidative stress was assessed by measuring glutathione (GSH) levels and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Supernatant protein concentrations were measured using a BSA kit (Beyotime Biotechnology). Levels of GSH and activities of SOD and GPx were measured using appropriate kits purchased from Beyotime Biotechnology according to the manufacturer’s instructions.
6
Quantifying Temporal Proteome Changes in RIMVECs
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RIMVECs were treated with IL-1α (final concentration of 10 ng/mL) at 37°C incubated in a 5% CO2 atmosphere for 0, 2, 4, and 8 h. The proteins of RIMVCEs were extracted by RIPA lysate buffer (Beyotime) and quantified by a BSA kit (Beyotime). The concentrations of sample proteins were detailed in Supplementary Table S1 . Two hundred microgram proteins were incubated in iTRAQ-4-plex kit (AB Sciex, PN: 4352135) proteolysis. The iTRAQ labeled, LC-MS/MS analysis and MALDI-TOF-TOF identifications were conducted by BIOMS company. Labels of 114, 115, 116, and 117 are represented 2, 4, 6, and 0 h treatment, respectively (Supplementary Table S1 ).
7
Quantitative Ferritin Assay in Mouse Testes
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Mouse testicular tissue (approximately 30 mg) was added to 300 μl of saline. The samples were then homogenized on ice and centrifuged at 200g for 10 min at 4°C. Following collection of the supernatant, standard products were prepared according to the instructions of the BSA kit (Beyotime Biotechnology Co., Ltd., Shanghai, China), and added to a 96-well microtiter plate. After incubation at 37°C for 30 min, the absorbance was measured at 562 nm to calculate the protein concentration. The cells were processed according to the operating procedure of the mouse ferritin ELISA kit (Zhenke Biotechnology Co., Ltd.). A microplate (Thermo Multiskan FC) was then used to measure the absorbance at a wavelength of 580 nm. Finally, ferritin concentration was calculated.
8
Western Blot Analysis of Ion Channels
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Western and IP lysis buffer (Beyotime) was used to lyse tissue and extract total protein. Lysate total protein concentrations were assayed with a BSA kit (Beyotime). Lysates were mixed with gel loading buffer, boiled, and separated by 12% polyacrylamide gel electrophoresis (20 μg protein per lane). Separated proteins were electrically transferred onto the nitrocellulose (NC) membranes. Membranes were blocked in buffer containing 5% skimmed milk powder overnight at 4ºC and then immunolabeled with a primary antibody [HCN2 (Bioss): 1:500; HCN4 (Bioss): 1:250; GAPDH (Santa Cruz, Santa Cruz, CA, USA): 1:500] at room temperature (RT) for 2 h. Membranes were washed 3 times with 1×TBST (10 min/wash), incubated in a corresponding secondary antibody solution at RT for 1 h, and washed 3 times with 1×TBST (10 min/wash). Immunolabeling was visualized by ECL chemiluminescent substrate (Millipore) and images acquired by the ChemiDoc-It HR 410 imaging system (Upland, CA).
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