Nuclear proteins were extracted using a nuclear extraction kit for mice (Beyotime Institute of Biotechnology, Beijing, China). For extracting total protein, cells were lysed in RIPA extraction buffer (Beyotime Institute of Biotechnology, Beijing, China), liver tissues were homogenized with a RIPA extraction buffer, incubated on ice for 2 h, and the lysates were centrifuged at 12000 g for 10 minutes at 4°C. Sample protein concentrations were determined by a BCA assay (BOSTER, Wuhan, China). Equal quantities of proteins from liver tissues were loaded and separated by 10% and 12% sodium dodecyl sulfate polyacrylaminde gel electrophoresis (SDS-PAGE). The proteins were subsequently transferred to a PVDF membrane (0.45 mm, Bio-Rad). The membranes were blocked with 5% non-fat milk, washed for three cycles of ten minutes, incubated with primary antibodies at 4°C overnight, and then incubated with the appropriate secondary antibody for 1 h at room temperature. Protein bands on the membrane were detected with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc., USA). Band density was quantified by ChemiDoc Quantity-One (Bio-Rad Laboratories) software. β-actin expression was used as the control reference. Lamin B1 was used as the nuclear protein control reference.
Ripa extraction buffer
Manufactured by Beyotime
Sourced in China
RIPA extraction buffer is a commonly used detergent-based lysis buffer designed for the extraction and solubilization of proteins from cells and tissues. It contains a mixture of ionic and non-ionic detergents, as well as other components that help preserve the native structure and function of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Tank transfer system, by Bio-Rad (5 mentions)
Enhanced chemiluminescence, by Merck Group (5 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Chemidoc quantity one, by Bio-Rad (2 mentions)
Bca assay, by Boster Bio (2 mentions)
Enhanced chemiluminescence ecl reagent, by Beyotime (2 mentions)
Bca protein kit, by Solarbio (2 mentions)
β actin, by Abcam (1 mentions)
Western lightning chemiluminescence reagent, by PerkinElmer (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
β actin antibody, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Rabbit anti inos, by Proteintech (1 mentions)
Rabbit anti cd206, by Proteintech (1 mentions)
14 protocols using ripa extraction buffer
1
Nuclear Protein Extraction and Analysis
2
Western Blot Analysis of Astrocyte C3
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Astrocytes or brain tissues were digested in RIPA extraction buffer (Beyotime, China). Protein samples were separated by 8% SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membranes (Millipore, United States) in tank transfer system (Bio-Rad, United States). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour, washed three times in TBST, and incubated overnight at 4°C with primary antibodies against C3 (1 : 1000 dilution, Abcam, USA) or β-actin (1 : 1000 dilution, Abcam, USA). After incubation with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1 : 10000 dilution, Da-UN, China), immunoreactive bands were detected by enhanced chemiluminescence (Millipore, United States). The protein bands were quantitatively analysed using ImageJ software 1.52a.
3
Protein Expression Analysis in Cell Lysates
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Cells were lysed in RIPA extraction buffer (Beyotime, Haimen, China) on ice for 30 min, then protein samples were separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membranes were probed with primary antibodies specific for FABP4, PPARγ, pRb, CDK1, CyclinB1, E2F1, P21, P18, phospho-C/EBPβ (T235 + T188), C/EBPβ, and β-actin, followed by incubating with secondary antibodies and then developed using Western Lightning Chemiluminescence Reagent (Perkin-Elmer Life Science, MA, USA).
4
Protein Extraction and Western Blot Analysis
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Cells were centrifuged at 500×g for 5 min and the supernatant was discarded. The cell pellet was re-suspended in cold RIPA extraction buffer (Beyotime Inc, 100 μl for 1 × 106 cells) in the presence of protease inhibitor cocktail and phosphatase inhibitor cocktail (Beyotime Inc). After staying on ice for 30 min, the lysate was sonicated for 30 s with 50% pulse. The lysate was centrifuged at 12,000×g for 15 min and the protein-containing supernatant was collected into a new tube. Extracted proteins were quantified using the Micro BCA™ Protein Assay Kit (Thermo Fisher). Each sample containing 10 μg protein was subjected to electrophoresis, transfer, antibody incubation, and chemiluminescent detection. The polyclonal ZFP335 antibody (TA345356, 1:1000) was obtained from OriGene Wuxi Biotechnology Co., Ltd. The JAK1 antibody (MA5-32780, 1:1000), AK3 antibody (MA5-15561, 1:1000), and β-Actin antibody (MA1-140, 1:2000) were purchased from Invitrogen. A Biospetrum 300 (UVP Ltd) was applied for imaging and signal quantification.
5
Quantitative Protein Expression Analysis
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Rat kidney tissues of the six groups were rapidly homogenized and then lysed in cold RIPA extraction buffer (Beyotime, Haimen, China) and then incubated in ice for 2 h. The lysate was centrifuged at 12000 rpm for 15 min at 4 °C and the supernatant was collected. The protein concentration was determined using BCA Assay (BOSTER, Wuhan, China). Equal amounts of protein solutions were mixed with Sodium dodecylsulfate sample buffer and incubated for 5 min at 98 °C before loading. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological blotting were performed on the basis of the method of Amersham Biosciences (Buckinghamshire, UK). Protein expression bands were detected by a chemiluminescent detection system (Syngen, Cambridge, UK) and analyzed with Chemidoc-Quantity-One (Bio-Rad Laboratories) software.
6
Western Blot Analysis of Protein Expression
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Cell precipitates were collected by conventional centrifugation, and cells were lysed on ice with RIPA extraction buffer (Beyotime, Shanghai, China) containing protease inhibitors. Fifteen minutes later, the proteins were collected by centrifugation at a low temperature and high speed, and the protein quantification and concentration were determined with a BCA protein kit (Solarbio, Beijing, China). Subsequently, the protein lysates were separated by SDS–PAGE and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. B-Actin expression was used as an internal control for normalization. The bands were detected with enhanced ECL chemiluminescence reagent (Beyotime). The details of the antibodies used in the experiments are listed in Supplementary Table S3
7
Proteomic Analysis of Lung Tissue
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Lungs were excised, immediately frozen at −80°C, and ground in liquid N2. Cold RIPA extraction buffer (Beyotime, Haimen, China) was added to the pulverized tissue and then the mixture was sonicated. Next, 1 mM phenylmethanesulfonyl fluoride (Beyotime), 2 mM ethylenediaminetetraacetic acid, 10 mM dithiothreitol, and protease inhibitor cocktails (Roche, Basel, Switzerland) were added, after which the mixture was centrifuged at 4°C and 30,000 × g for 15 min. The supernatant was collected and added to five volumes of cold acetone containing 10% (v/v) trichloroacetic acid, thoroughly mixed, and incubated at −20°C overnight. The mixture was centrifuged again at 4°C and 30,000 × g and the supernatant was discarded. The precipitate was then washed three times with chilled acetone, dissolved in RIPA buffer, and air-dried. Proteins were quantified with a BCA kit (Thermo Fisher Scientific, Waltham, MA, United States), after which 300 μg of total protein was mixed with sequencing-grade trypsin (Promega, Madison, WI, United States) at an enzyme-to-protein ratio of 1:50 and incubated at 37°C for 16 h. Peptides obtained from the digestion were dried by vacuum centrifugation.
8
Protein Extraction and Western Blot Analysis
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Cells were lysed on ice for 20 min using RIPA extraction buffer (Beyotime, Shanghai, China) containing protease inhibitor. Proteins were collected and the concentrations were determined by BCA protein kit (Solarbio, Beijing, China). Then, protein lysates were separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Darmstadt, Germany). The membranes were blocked with 5% defatted milk for 1 h and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. The β-Actin expression was used as internal control for normalization. The brands were detected by enhanced ECL chemiluminescence reagent (Beyotime). Details of the antibodies used in experiments were listed in Additional file 1 : Table S2.
9
Western Blot Analysis of Neuroinflammatory Markers
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BV2 cells and mouse spinal cord L4‐5 segment tissues were digested in RIPA extraction buffer (Beyotime, China). Protein samples were separated by 10% SDS‐PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, United States) in tank transfer system (Bio‐Rad, United States). Membranes were blocked with 5% nonfat milk in buffer containing 0.1% Tween‐20 (TBST) for 1 h, washed three times in TBST, and incubated overnight at 4°C with primary antibodies including rabbit anti‐Wnt5a (1:1000 dilution; Proteintech; USA; 55,184‐1‐AP), rabbit anti‐CaMKII (1:1000 dilution; Proteintech; USA; 13,730‐1‐AP), rabbit anti‐NFAT (1:1000 dilution; Proteintech; US; 22,023‐1‐AP), rabbit anti‐GAPDH (1:10000 dilution; Proteintech; USA; 10,494‐1‐AP), rabbit anti‐iNOS (1:1000 dilution; Proteintech; US; 18,985‐1‐AP), and rabbit anti‐CD206(1:1000 dilution; Proteintech; US; 18,704‐1‐AP). After incubation with the HRP‐conjugated goat anti‐rabbit IgG secondary antibody (1:10000 dilution, Da‐UN, China), immunoreactive bands were detected by enhanced chemiluminescence (Millipore, United States). The protein bands were quantitatively analyzed using ImageJ software 1.8.0 (National Institutes of Health, Bethesda, MA, USA).
10
Western Blot Analysis of Apoptosis Proteins
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Western blotting analysis was used to assess the expression of apoptosis-related protein of the LSG. The total protein was extracted from the LSG tissue. The LSG tissues were lysed in RIPA extraction buffer (Beyotime, China) and then centrifuged at 12,000 rpm for 10 min at 4 ºC. The total protein concentration was measured by BCA Protein Assay Kit (TIANGEN, China). The LSG proteins were separated by electrophoresis in 12% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed with antibodies against β-catenin (1:1000; Abcam), Bcl-2 (1:1000; CST), Bax (1:1000; CST), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH 1:3000; Affinity). Next, the membranes were followed by incubation with secondary antibodies, and finally were exposed with ECL reagent (Proteintech, China), and scanned by the Imaging System. The content of β-catenin, Bcl-2 and Bax were normalized to GAPDH. A minimum of three independent WB experiments were performed, and the Image-lab 4.0.1 software was used for analysis.
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