Total protein was extracted from SD rat adipose tissue using Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Dalian Meilun Biotechnology Co., LTD., Dalian, China) containing protease and phosphatase inhibitor (Beyotime, Shanghai, China). Protein samples were diluted using loading buffer and denatured at 98 °C for 5 min. The protein sample was electrophoresed on the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at a concentration of 5–15% (v/v), and the isolated protein was subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp., Billerica, MA, USA). The PVDF membrane was blocked with 5% skimmed milk powder. After blocking, the PVDF membrane was incubated overnight at 4 °C in diluted primary antibody and then incubated with goat anti-rabbit IgG/horseradish peroxidase (HRP) secondary antibody for 1 h (Biosynthetic Biotechnology Co., Ltd., Beijing, China). Primary antibodies include UCP1, PRDM16, PGC-1α, P38-MAPK, ATF2, and β-actin (Abcam plc. shanghai, China) (Table S4 ). Gray analysis of Western blots was performed by Image J 1.48V software (National Institutes of Health, USA).
Protease and phosphatase inhibitor
Manufactured by Beyotime
Sourced in China
Protease and phosphatase inhibitors are laboratory reagents used to prevent the degradation of proteins and the dephosphorylation of phosphoproteins in biological samples. These inhibitors work by blocking the enzymatic activity of proteases and phosphatases, which can otherwise break down or modify the target proteins during sample processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (48 mentions)
Pvdf membrane, by Merck Group (42 mentions)
Ripa lysis buffer, by Beyotime (34 mentions)
Bca protein assay kit, by Beyotime (20 mentions)
Bca kit, by Beyotime (10 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (5 mentions)
Gapdh, by Abcam (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
β actin, by Proteintech (4 mentions)
Loading buffer, by Beyotime (4 mentions)
Pvdf membrane, by Beyotime (4 mentions)
Ab8245, by Abcam (4 mentions)
Amersham imager, by Cytiva (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (4 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
β actin, by Abcam (3 mentions)
169 protocols using protease and phosphatase inhibitor
1
Protein Extraction and Western Blot Analysis
2
Retinal Protein Quantification After Injury
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Total protein was obtained from retinal tissue on the fifth day after RIR injury. Radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China) was added into dissected retinas. Protein samples were separated by a 10-12% SDS-PAGE gel (Beyotime Biotechnology, Shanghai, China) and transferred to polyvinylidene difluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA). The PVDF membranes were blocked with 5% BSA in tris-buffered saline with Tween 20 (TBST) for 2 hours at room temperature and incubated at 4 ℃ overnight with primary antibodies. Primary antibodies consisted of goat anti-LCN2 antibody (1:1,000; Cat#AF1857; R&D Systems, Minneapolis, USA), GPX4 (1:1,000; Cat#67763-1-lg; Proteintech), and β-tubulin (1:2,000; Cat#2146; CST). Subsequently, membranes were incubated with corresponding secondary antibodies (1:5,000) for 2 hours at room temperature, and the blots were shown with an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Waltham, MA, USA) and visualized with an Image Lab imaging system (Uvitec, Cambridge, UK). The protein levels were normalized to tubulin and expressed relative to the WT or control.
3
Western Blot Analysis of Endothelial Cell Stress
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HCMECs were collected and lysed with RIPA lysis buffer [50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS] and protease and phosphatase inhibitor (Beyotime, China) after treatment. The total protein concentration was determined using the bicinchoninic acid (BCA) reagent (Beyotime, China). Protein samples (50–100 μg each) were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After gel electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane. The membranes were trimmed according to the protein sample width, and all proteins were strictly retained during the modification process. After blocking in 5% nonfat milk, the membranes were probed with primary antibodies for GRP78 (1:1000), p-eNOSSer1177 (1:1000), eNOS (1:1000), PERK (1:500), p-eIF2α (1:1000), eIF2α (1:500), IRE-1 (1:1000), p-JNK (1:500), JNK (1:1000), ATF6 (1:1000), or β-actin (1:5000) and subsequently labeled with horseradish peroxidase-conjugated secondary antibody. The bands were visualized by the enhanced chemiluminescence reagent (Millipore, US) and autoradiography, and protein gray analysis was proceeded with Quantity One (BioRad, Canada). All results were representative of three independent experiments.
4
Immunoprecipitation and Western Blot Analysis
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Samples were lysed in NP-40 Lysis Buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitor (Beyotime Biotechnology). Cell lysates were collected and incubated with protein A/G Plus-Agarose (Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C for 4 h. The suspension was then incubated with the corresponding primary antibodies together with protein A/G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C overnight. Agarose were then washed five times with NP-40 Lysis Buffer (Beyotime Biotechnology) and prepared for Western blot analysis. When appropriate, cells were treated with 10 μM MG-132 (Selleck Chemicals, Houston, TX, USA).
5
Proteomic Analysis of HUMSC-sEVs and HCECs
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HUMSC-sEVs and HCECs were lysed in lysis buffer containing protease and phosphatase inhibitor (Beyotime Institute of Biotechnology, China). Proteins were separated by electrophoresis after loading onto polyacrylamide gel and then transferred to the PVDF that was incubated with primary antibodies against phospho-Akt (4060s, CST), PTEN (9188, CST), CD9 (ab92726, Abcam), CD61 (ab59479, Abcam), and CD81 (00679767, Invitrogen) overnight at 4°C after blocking with 5% nonfat milk, followed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibody. Proteins were detected with a Western blot analysis system.
6
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from colonic tissues and LGG-EVs using RIPA buffer containing protease and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). The equal quantity of protein was loaded into gels, and then transferred onto PVDF membranes (Thermo Fisher Scientific). Proteins were quantified by Image J software (NIH, Bethesda, MD). Antibodies against TSG101 (ab225877), p65 (ab16502), p-p65 (ab76302), NLRP3 (ab210491), ASC (ab180799) were purchased from Abcam (Cambridge, UK). β-actin (GB12001) was purchased from Servicebio (Wuhan servicebio technology CO., LTD, Wuhan, China).
7
Western Blot Analysis of Protein Samples
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The cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor (Beyotime, Shanghai, China), and the protein concentration of the samples was determined with a BCA kit (Beyotime Biotechnology, Beijing, China). Next, 10 µg of protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membranes were sealed for 1 h with 5% non-fat milk, followed by incubation with primary antibodies (Table 1 ) at 4 °C overnight. The membranes were then incubated with suitable HRP-conjugated secondary antibodies (Boster Biological Technology, Wuhan, China) at an ambient temperature for 1 h. The bands were visualized with Chemidoc XRS Gel Imaging using an ECL Substrate (Millipore, Burlington, MA, USA). Finally, membranes were stripped and re-probed. Each band was quantified by densitometry using Image J, β-actin used for normalization [46 (link)].
8
Western Blot Analysis of Glioma Signaling
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The glioma cells were lysed using RIPA buffer with Protease and Phosphatase inhibitor (Beyotime). The total protein concentration was examined using a BCA kit. Equal amounts of total protein (30 μg) were electrophoresed in 10%–12% SDS‐PAGE and electro‐transferred onto NC membrane (GE). After blocking with 5% non‐fat dry milk in TBST for 1 h at room temperature (RT), the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with HRP‐labeled secondary antibodies (Proteintech) for 1 h at RT. An enhanced chemiluminescence reagent (Millipore) was used to detect protein expression value. The relative quantity of proteins was analyzed using the Image J software. The primary antibodies used are as follows: anti‐EZH2 (1:2000, #66476‐1‐Ig, Proteintech), anti‐VEGFA (1:1000, #66828‐1‐Ig, Proteintech), anti‐VEGFR2 (1:200, #sc‐6251, Santa‐Cruz Technology), anti‐PhosphoTyr1175‐VEGFR2 (1:1000, #2478, CST), anti‐AKT (1:1000, #9272, CST), anti‐ PhosphoThr308‐AKT (1:1000, #13038, CST), anti‐ERK1/2 (1:1000, #4695, CST), anti‐PhosphoThr202/Tyr204‐ERK1/2 (1:1000, #4370, CST), anti‐GAPDH (1:5000, #60004‐1‐Ig, Proteintech), and anti‐β‐tubulin (1:5000, #10094‐1‐AP, Proteintech).
9
Western Blot Analysis of TGR5 and NF-κB Signaling
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Tissues and cells were lysed using RIPA buffer supplemented with 1 mM PMSF and protease
and phosphatase inhibitor (Beyotime, Shanghai, China). Lysates were incubated on ice and
cleared by centrifugation at 12 000 × g for 10 min at 4°C. Proteins were
separated by 10% denaturing polyacrylamide gel electrophoresis and transferred onto
polyvinylidene difluoride membranes. After blocking in 5% nonfat milk, the membranes were
exposed to specific primary antibodies against TGR5 (Abcam), β-catenin, p-β-catenin,
p-Akt, p-Gsk3β, p-NF-κB p65, TLR4 and β-actin (Cell Signaling Technology) at a
concentration of 1:1000. Membranes were then washed and exposed to peroxidase-conjugated
secondary antibodies (Cell Signaling Technology). Immunoblots were imaged using a medical
film processor (SRX-101A, Konica Minolta Medical & Graphic, Inc., NY, USA).
and phosphatase inhibitor (Beyotime, Shanghai, China). Lysates were incubated on ice and
cleared by centrifugation at 12 000 × g for 10 min at 4°C. Proteins were
separated by 10% denaturing polyacrylamide gel electrophoresis and transferred onto
polyvinylidene difluoride membranes. After blocking in 5% nonfat milk, the membranes were
exposed to specific primary antibodies against TGR5 (Abcam), β-catenin, p-β-catenin,
p-Akt, p-Gsk3β, p-NF-κB p65, TLR4 and β-actin (Cell Signaling Technology) at a
concentration of 1:1000. Membranes were then washed and exposed to peroxidase-conjugated
secondary antibodies (Cell Signaling Technology). Immunoblots were imaged using a medical
film processor (SRX-101A, Konica Minolta Medical & Graphic, Inc., NY, USA).
10
Western Blot Analysis of Protein Lysates
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Protein lysates were extracted using RIPA Lysis Buffer supplemented with protease and phosphatase inhibitor (Beyotime Biotechnology), and measured with a BCA assay. Cell lysates were separated on SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% milk for 1 h, and then incubated with the corresponding primary antibodies at 4 °C overnight. Next, membranes were incubated with secondary antibodies at room temperature for 1 h, and finally visualized using an enhanced chemiluminescence luminescence reagent (Meilunbio). ImageJ software was used to calculate the intensities of the bands. Primary antibodies used are as follows: β-actin (Sigma-Aldrich; SAB1305554), FLAG (Sigma-Aldrich; F1804), NLRP6 (Sigma-Aldrich; SAB1302240), and Rev-erbα (Proteintech; 14506-1-AP).
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