Cells were treated with doxorubicin and fulvestrant alone or in combination for 24 h. Cellular protein was isolated with a protein extraction buffer (Beyotime). Protein concentrations were measured with the BCA protein assay kit (Pierce). Equal amounts (40 µg/lane) of proteins were fractionated on 10–12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the desired primary antibodies, respectively. After washing with PBS containing 0.1% (v/v) Tween-20, the membranes were incubated with anti-mouse or anti-rabbit IgG coupled to HRP second antibodies for 2 h at room temperature followed by enhanced chemiluminescent staining using the ECL system. β-actin was used for normalization of protein loading.
Protein extraction buffer
Manufactured by Beyotime
Sourced in China
Protein extraction buffer is a solution designed to facilitate the extraction and isolation of proteins from biological samples. It contains a combination of chemical agents that disrupt cell membranes and denature proteins, allowing for the efficient release and solubilization of proteins from the sample. The buffer's composition is formulated to maintain the structural integrity and functionality of the extracted proteins, enabling their subsequent analysis and purification.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (3 mentions)
Nucleoprotein extraction kit, by Sangon (3 mentions)
Hrp conjugated affinipure goat antibodies igg, by Boster Bio (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Electrochemiluminescence reagents, by Bio-Rad (2 mentions)
Anti e cadherin, by Proteintech (2 mentions)
Anti gapdh, by Bioworld Technology (2 mentions)
Bca protein assay kit, by Tiangen Biotech (2 mentions)
Pvdf transfer membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by CWBIO (1 mentions)
Imagej software, by GE Healthcare (1 mentions)
Ab11419, by Abcam (1 mentions)
Ab203119, by Abcam (1 mentions)
Ab37073, by Abcam (1 mentions)
Ab55878, by Abcam (1 mentions)
Ab81785, by Abcam (1 mentions)
Enhanced chemiluminescence solution, by Merck Group (1 mentions)
Ecl reagent, by Share-Bio (1 mentions)
28 protocols using protein extraction buffer
1
Protein Analysis of Doxorubicin and Fulvestrant
2
Revealing ER Stress and Autophagy Markers
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Following the desired treatments, CHO cells were washed and harvested on ice, and then lyzed with ice-cold protein extraction buffer (Beyotime, Nanjing, China). The protein concentration was determined using BCA protein assay kit (ComWin, Beijing, China). Equal amounts of protein were loaded into gel wells, separated by electrophoresis on SDS-polyacrylamide gels and transferred onto PVDF transfer membrane (Millipore, Bedford, MA). The transferred blots were blocked with blocking agents (5% w/v BSA and 0.05% Tween-20 in TBS) for 1 h at room temperature. Blots were then incubated in a certain proportion of specific primary antibodies: anti-GRP78 (ab25192 Rabbit monoclonal, Abcam), anti-ATF-6 (ab203119 Rabbit polyclonal, Abcam), anti-PERK (C33E10 Rabbit polyclonal, Abcam), anti-IRE1 (ab37073 Rabbit polyclonal, Abcam), anti-CHOP (ab11419 Mouse monoclonal, Abcam), anti-Beclin1 (ab55878 Rabbit polyclonal, Abcam), anti-LC3B (ab81785 Rabbit polyclonal, Abcam) (1:1000) overnight at 4°C. These membranes were rinsed 3 times for 10 min each with TBS-Tween (Sigma, St. Louis, MO, USA) and incubated with HRP conjugated secondary antibodies (1:5000) (CWBio, Beijing, China). After washing, protein bands were visualized by an enhanced chemiluminescence detection kit (ComWin, Beijing, China). Each band density was quantified using Image J software (GE Healthcare, Piscataway, New Jersey, USA).
3
MicroRNA-155 Regulation in VSMCs
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VSMCs were treated with 50 µM DHA and transfected with the microRNA-155 mimics and inhibitors before harvesting for protein isolation. Proteins were isolated in the protein extraction buffer (Beyotime Institute of Biotechnology). The protein concentration was measured by BCA method. Protein samples (20 µg/lane) were separated by SDS-PAGE on 10% gels and transferred to a nitrocellulose membrane. The membranes were incubated with 5% fat-free milk in TBS with Tween® 20 for 1 h at room temperature and with primary antibodies overnight at 4°C. The PVDF membranes were washed with TBST buffer and incubated with peroxidase-conjugated specific secondary antibodies from Abcam [Ki67 (1:5,000; cat. no. ab15580), Socs1 (1:4,000; cat. no. ab62584) and actin (1:3,000; cat. no. ab6276)] with shaking for 1 h. The enhanced chemiluminescence solution (Sigma-Aldrich; Merck KGaA) was the prepared in a dark room, and the exposure time of the film was determined according to the chemiluminescence intensity. Densitometric analysis was performed using Image Lab software (version 3.0; Bio-Rad Laboratories, Inc.).
4
Western Blot Analysis of Spinal Cord Proteins
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Western blotting was carried out as previously described [20 (link)]. Proteins were extracted from cells with the protein extraction buffer (Beyotime, Shanghai, China). Spinal cord samples (epicenter ± 0.3 cm) were collected and homogenized with 10 to 15 strokes (3–4 s/stroke) using a homogenizer and plastic pestle with protein extraction buffer. Subsequently, protein concentration was determined using the Bradford method, and equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with the following corresponding primary antibodies overnight at 4 °C followed by blocking with 5% skimmed milk or 5% Bovine Serum Albumin (BSA): anti-β-actin (1:1000), anti-MSR1 (1:1000), anti-cleaved-Caspase-3 (1:1000), anti-Bcl2 (1:1000), anti-Bax (1:1000), anti-IκBα (1:1000), and anti-pIκBα (1:1000). After incubating with species-matched secondary antibodies (1:10000), ECL reagents (Share-bio, Shanghai, China) were used to develop bands and the density of protein was accessed by the ImageJ (National Institutes of Health, Bethesda, MD, USA).
5
BAFF-R Signaling Pathway Analysis
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Major reagents included recombinant human BAFF (310–13, Peprotech, Rocky Hill, NJ, USA);BAFF-R antibody for flow cytometry and western blot (14–9117, eBioscience, San Diego, CA, USA); BAFF-R Fc chimera (1162-BR-050, R&D Systems, Minneapolis, MN, USA); phospho-Akt (2965) and Akt antibodies (9272, Cell Signaling Technology, Danvers, MA, USA); phospho-Erk (Thr202/Tyr204) (9106) and Erk antibodies (9102, Cell Signaling Technology); phospho-MAPKp38 (Thr180/Tyr182) (9211) and MAPKp38 antibodies (9212, Cell Signaling Technology); phospho-NF-κB p65 (Ser536) (3033) and NF-κB p65 antibodies (4764, Cell Signaling Technology); NF-κB p100 antibody(BS1247, Bioworld, China); Na+/K+-ATPase antibody (BS4259, Bioworld); carboxyfluoresceinsuccinimidyl ester (CFSE) (C34554, Life Technologies, Carlsbad, CA, USA); reverse transcription (RR014A) and real-time PCR kits (DRR036A, TAKARA, Shiga, Japan); TRIzol (15596–18) and DNaseI (AM2235, Life Technologies); protein extraction buffer (P0013, Beyotime, China); MTS Proliferation Assay kit (G3582, Promega, Madison, WI, USA); and a membrane protein extraction kit (89842, Pierce, Thermo Scientific, Waltham, MA, USA).
6
Comprehensive Western Blotting Protocol
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Western blotting was carried out as previously described 27 (link)-29 (link). The protein extraction buffer (Beyotime, Shanghai, China) or nucleoprotein extraction kit (Sangon Biotech, C500009) was used to extract total or nuclear cellular proteins according to the manufacturer's instructions. Further, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk or 5% Bovine Serum Albumin, the membrane was probed with the following primary antibodies: anti-β-actin (1:2000), anti-Histone H3 (1:1000), anti-MSR1 (1:1000), anti-AKT (1:1000), anti-p-AKT (1:1000), anti-GSK3β (1:1000), anti-p-GSK3β (1:1000), anti-mTOR (1:1000), anti-p-mTOR (1:1000), and anti-β-catenin (1:1000). The species-matched secondary antibodies were used (1:10000), and the bands were detected by the Odyssey imaging system (LI-COR, Lincoln, NE, USA).
7
Western Blotting of Neuronal Proteins
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Western blotting was carried out as previously described [25 (link)]. Briefly, the cells and brain tissues were homogenized in a protein extraction buffer (Beyotime, China). BCA protein assay was used to determine protein concentration (Beyotime, China). They were incubated overnight with primary antibodies (anti-NR2B, anti-ERK, anti-p-ERK, anti-CREB, anti-p-CREB, anti-BDNF, and anti-β-actin) at 4°C, followed by secondary antibodies for 2 h at ambient temperature. The films were captured using the ECL chemiluminescence system. Protein bands were quantified using ImageJ software (Version 1.46).
8
Protein Extraction and Western Blot Analysis
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Two hundred milligrams of plant tissue were collected and placed into a 2 ml grinding tube (Jingxin). After grinding plant tissues at low temperature, 1 ml of protein extraction buffer (Beyotime) was added, after which the contents were shaken at 4 °C for 30 min, followed by centrifugation at 13,000g and 4 °C for 15 min. The centrifuged supernatant was homogenized with a bicinchoninic acid kit (CWBIO), mixed with an equal volume of 2× SDS loading buffer and denatured at 100 °C for 8 min. The proteins were resolved using 10 to 20% SDS‒PAGE gels (Genescript). The Western blot–specific antibodies used included anti-GFP (ABclonal), anti-HA (CST), anti-Actin (ABclonal), anti-SWP1 (ABclonal), and HRP goat anti-rabbit immunoglobulin G (H + L) (ABclonal).
9
Protein Expression Analysis in Transgenic Zebrafish
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Proteins were extracted from the lens, lens-excluded eye tissues, and eyes-excluded body tissues of zTg (transgenic zebrafish) and WT zebrafish at 15 dpf using protein extraction buffer (Beyotime). The antibodies used in this study were anti-Cre recombinase (15036S, Cell Signaling Technology, Danvers, United States) and anti-Gapdh (10,494-1-AP, Proteintech, Wuhan, China) at a dilution of 1:1,000. The extracted proteins were separated using 10% SDS–PAGE (sodium dodecyl sulfate-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% dry milk/phosphate buffer solution and Tween-20 (PBST), the membranes were incubated in a solution with appropriate primary antibodies. Next, they were incubated with a secondary antibody conjugated with horseradish peroxidase. The signal was developed in the ECL buffer and detected using a charge coupled device (CCD)-camera-based imager (Amersham Imager 680, Amersham, CA; GE).
10
Subcellular Protein Fractionation and Western Blot
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Whole-cell lysates or nuclear protein was extracted using a protein extraction buffer (Beyotime, Shanghai, China) or nucleoprotein extraction kit (Sangon Biotech, C500009), respectively. Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose (NC) membranes using standard methods. Primary antibodies used as follows: MPC1 (ab74871, Abcam), β-catenin (ab32572, Abcam), lamin A/C (ab8984, Abcam), and GAPDH (ab9485, Abcam). Species-specific secondary antibodies used as follows: IRDye 680 Goat anti-Mouse IgG (LI-COR) and IRDye 800 Goat anti-rabbit IgG (LI-COR).
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