Lung tissues or PASMCs were lysed in RIPA buffer containing phosphatase and protease inhibitors (ST505, Beyotime, China) on ice and centrifuged at 12,000 × rpm for 20 min at 4 °C. Protein concentrations were detected using a BCA protein assay kit (Beyotime, China), and equal amounts of protein (40 μg each lane) were subjected to SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). Membranes were incubated by primary antibodies for 18 h, including AT1R (1:4000, ab124734, Abcam, USA) and α-tubulin (1:5000, T5168; Sigma, St. Louis, MO, USA), followed by HRP-conjugated secondary antibodies for 2 h at room temperature. ECL was used to detect the immunoreactive bands, and blots densitometry was analysed by ImageJ software.
St505
Manufactured by Beyotime
Sourced in China
The ST505 is a digital pH/mV/temperature meter designed for accurate and reliable measurements. It features a large LCD display, automatic temperature compensation, and the ability to measure pH, mV, and temperature. The meter is compact, portable, and easy to use.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab8245, by Abcam (3 mentions)
Ipvh00010, by Merck Group (3 mentions)
Protein quantification kit, by Beyotime (2 mentions)
P1005, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
β actin, by Merck Group (2 mentions)
Orai1, by Abcam (2 mentions)
T5168, by Merck Group (1 mentions)
Ab124734, by Abcam (1 mentions)
P0011, by Beyotime (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab76198, by Abcam (1 mentions)
Ab76199, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
28 protocols using st505
1
AT1R Expression in Lung Tissues
2
Western Blotting Quantification Protocol
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Western blotting was performed as previously described [20 ]. Proteins were collected using RIPA buffer (P0013B, Beyotime, China), protease inhibitors (p1005, Beyotime, China), and phenylmethylsulfonyl fluoride (ST505, Beyotime, China), quantified by a protein quantification kit (P0011, Beyotime, China), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, electrophoresis was conducted to transfer the proteins into a PVDF membrane (160-0184, Bio-Rad, USA). The membrane was blocked by 5% dried skimmed milk powder for 1 h and then incubated with antibodies against Akt (1: 500, 56kDa, ab8805, Abcam, UK), phosphorylated (p)-Akt (1: 1000, 56kDa, ab38449, Abcam, UK), endothelial nitric oxide synthase (eNOS) (1: 1000, 133kDa, ab76198, Abcam, UK), p-eNOS (1: 500, 140kDa, ab76199, Abcam, UK), and GAPDH (1: 1000, 36kDa, ab8245, Abcam, UK). After incubation for 24 h, the membrane was washed by 1% Tris-buffered saline with Tween 20 and incubated with goat anti-rabbit secondary antibody (1: 10 000, ab205718, Abcam, UK) or goat anti-mouse secondary antibody (1: 10000, ab6789, Abcam, UK). An ECL luminescence kit (PE0010, Solarbio, China) and a gel imaging system (FluorChem FC3, Alpha, USA) were used to expose the membrane and visualize the protein bands, respectively. ImageJ2x (Rawak Software, Germany) was used to analyze the results.
3
Western Blot Analysis of MAPK Signaling
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PASMCs were lysed in radio‐Immunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitors (ST505; Beyotime) on ice and centrifuged at 12,000 rpm for 20 min at 4°C. Protein concentrations were determined using a BCA assay (Beyotime) and equal amounts of protein (40 μg each lane) were subjected to SDS‐PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were blocked with 5% fat‐free milk, and incubated overnight at 4°C with primary antibodies including anti‐MKK3 (AF6327; Affinity Biosciences), anti‐MKK6 (AF7820; Affinity Biosciences), anti‐P‐38 (AF6456; Affinity Biosciences), and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (ab181602; Abcam). After washing with PBST five times for 5 min each, the membranes were incubated with secondary antibodies for 1 h at room temperature. Target bands were quantified using a Tanon‐5200 Image Analyzer.
4
Immunoblotting of Cardiac Ion Channels
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Cardiac tissues were homogenized, and the cultured cells were ultrasonicated in radioimmunoprecipitation assay buffer (P0013C; Beyotime) with 1 mmol/L phenylmethylsulfonyl fluoride (ST505; Beyotime). After protein concentrations were measured, the samples were heated at 99°C for 5 minutes. After electrophoresis on 10% SDS‐PAGE gels, proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% BSA in TBST buffer (10 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, and 0.05% Tween‐20) for 2 hours at room temperature. The following primary antibodies were used: anti‐rabbit Kcna2 (1:200; Alomone Labs), anti‐rabbit kvβ1 (1:1000; Abcam, Cambridge, UK), and anti‐GAPDH (1:1000; Cell Signaling Technology, Boston, MA) at 4°C overnight. Proteins were detected using horseradish peroxidase–conjugated anti‐rabbit (Biosynthesis, Beijing, China) or anti‐mouse (Santa Cruz, Dallas, TX) antibodies and were visualized using the ChemiDoc XRS Imaging System (Bio‐Rad, Hercules, CA). Blot intensity was quantified via densitometry.
5
Western Blot Analysis of GPX4 and FTH
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Lysis buffer (Beyotime, P0013B) containing 1% protease inhibitor (Beyotime, ST505) was used to extract protein. The protein assay kit (Beyotime, P0010S) was used to detect protein concentration. The equal amount of protein was separated by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. The membranes were blocked with blocking reagent (Beyotime, P0252) for 1 hr followed by incubation with primary antibody GPX4 (CST, #52455), ferritin heavy chain (FTH) (CST, #4393S), and Tubulin (Beyotime, AF1216) at 4°C overnight. Then, the membranes were incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (Beyotime, A0208) for 1 hr at room temperature. The membranes were observed and photographed by Bio-Rad chemiluminescence imaging system after enhanced chemiluminescence solution (Beyotime, P0018S) was exposed. The image gray value was analyzed quantitatively by Image J 1.8.
6
Western Blot Analysis of Apoptosis and Signaling
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Tumor tissues were homogenized using TissueLyser II (Qiagen) according to manufacturer' instructions. Protein from tissues and CCA Cells were isolated using RIPA buffer (P0013B, Beyotime, China) with phosphatase inhibitor (ST505, Beyotime, China). 20 μg of protein from each sample used for the 10% SDS-PAGE and then transferred to PVDF membrane (IPVH00010, Millipore, Germany). After transfer and blocking, membranes were incubated with following antibodies, Bax (1:1000, 60267, proteintech, China), Bcl-2 (1:1000, 60178, proteintech, China), caspase-3 (1:1000, 14220, CST, USA), Cleaved-Caspase3 (1:1000, 9664, CST, USA), MMP-9 (1:1000, 3852, CST, USA), Vimentin (1:1000, 3932, CST, USA), PPP1R12A (1:1000, ab32519, Abcam, UK), phospho-PI3K (1:1000, AP0854, Abclonal, China), phospho-Akt (1:1000, 4060, CST, USA), phospho-p38 (1:1000, 4511, CST, USA), phospho-JNK (1:1000, 9255, CST, USA), phospho-ERK1/2 (1:1000, 4370, CST, USA), and GAPDH (1:1000, ab8245, Abcam, UK). The membranes were washed using TBST and then incubated with correspondent secondary antibody. DAB Horseradish Peroxidase Color Development Kit was used for development (P0018S, Beyotime, China). The statistical gray value of each strip, and then calculate the relative expression.
7
Western Blot Analysis of Inflammatory Proteins
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Lung tissues and RAW264.7 cells were lysed in RIPA lysis buffer (P0013B,Beyotime) supplemented with protease inhibitor (ST505, Beyotime) and phosphatase inhibitor (P1050, Beyotime), and the protein concentration in each sample was determined using the BCA assay. Samples were denatured, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The non-specific membrane binding sites were blocked with 5% bovine serum albumin (BSA) for 1 h. Membranes were then incubated at 4°C overnight with primary antibodies (all diluted 1:1000) against the following proteins: IL-17 (ab79056, Abcam), MCP-1 (catalog no. 2029, CST),p38 MAPK (catalog no. 9212, CST), and phosphorylated (p)-p38 MAPK (catalog no. 4511, CST). Blots were washed several times with PBS buffer, then incubated for 1 h with goat anti-rabbit IgG secondary antibody (diluted 1:15000; ab96899, Abcam). The bands were visualized using a fluorescent scanner. As an internal reference, GAPDH was immunostained using an antibody (1:1000; catalog no. 5174, CST).
8
Western Blot Analysis of Muscle Proteins
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Porcine satellite cells or muscle tissues were lysed in RIPA buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (PMSF) (ST505, Beyotime, China) to acquired total protein. Approximately 30 μg of protein of each sample was loaded on SDS-PAGE and transferred to PVDF membranes (IPVH85R, Millipore, United States). After blocking with 5% non-fat milk, the proteins in membranes were subjected to immunoblotting analysis with primary and secondary antibodies. Antibodies used in this study were as follows: anti-H3K27me3 (17-622, Millipore, United States), anti-beta-tubulin (GB11017, Servicebio, China), anti-MYOD (sc-760, Santa Cruz Biotechnology, United States), anti-MYH4 (A15293, ABclonal, United States), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (SA00001-2, Proteintech, United States). The membranes were developed with ECL (WBULS0500, Millipore, United States) for visualization. ImageJ software was used to determine the bands’ signal intensities. The density value of each band was normalized by corresponding beta-tubulin density value.
9
Protein Extraction and Western Blot Analysis
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Cells were lysed using RIPA buffer containing 1% Phenylmethanesulfonyl fluoride (PMSF, ST505, Beyotime, Shanghai, China) and 2% phosphatase inhibitor. The total protein concentration was quantified using the Bicinchoninic Acid Protein Assay Kit (ST505, Thermo Fisher Scientific, Waltham, MA, United States). Next, protein samples (30 mg) were separated using 10% SDS-PAGE (PG212, EpiZyme, Shanghai, China). Then, the proteins were transferred to PVDF membranes, followed by blocking with 5% BSA (A8020, Solarbio, Beijing, China) at room temperature for 1 h. Afterwards, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were washed with PBST and incubated with a goat anti-mouse or goat anti-rabbit secondary antibody for 1 h at room temperature. Finally, the protein bands were visualized with ECL (Millipore) and quantified with ImageJ software (National Institutes of Health) the manufacturer’s instructions.
10
Immunoprecipitation of DNALI1 from Sperm and Testes
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To conduct IP, we accumulated 8 ml of human sperm, and rinsed thrice with PBS, prior to lysis in RIPA buffer (P0013B, Beyotime) with phosphatase inhibitor cocktail A,50X (P1082, Beyotime) and phenylmethanesulfonyl fluoride (ST505, Beyotime). Both human sperm and mice testes (200 mg) underwent homogenization in 2 ml lysis buffer on ice for 40 min, prior to centrifugation at 12,000 × g for 20 min at 4 °C. The supernatant was saved in a new tube for IP analysis as follows: First, it underwent ON incubation with anti-DNALI1 at 4 °C, followed by treatment with protein A/G (Bimake, B23202) beads for 2 h at 4 °C. Subsequently, the precipitants were twice rinsed in IP buffer (20 mM Tris, pH 7.4, 2 mM EGTA and 1% NP-40), and the immune complexes were eluted with sample buffer and 1% SDS for 10 min at 95 °C, prior to analysis via immunoblotting or mass spectrometry (MS).
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