Cells were isolated using the protein lysis buffer (Beyotime, China) according to the manufacturer’s instructions. The primary antibodies used in our study were an anti-PRDX6 antibody (abcam, 1:1000), anti-RARA antibody (abcam, 1:1000), anti-β-Actin antibody (abcam, 1:2000) overnight at 4°C. β-Actin was used as the reference control.
Protein lysis buffer
Manufactured by Beyotime
Sourced in China, United States
Protein lysis buffer is a solution used to extract and solubilize proteins from cells or tissues. It is a fundamental tool in biochemistry and molecular biology research for protein analysis and purification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (15 mentions)
Bca protein assay kit, by Beyotime (11 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Polyvinylidene fluoride membrane, by Merck Group (7 mentions)
Ecl kit, by Thermo Fisher Scientific (6 mentions)
Bca kit, by Beyotime (5 mentions)
Ab181602, by Abcam (4 mentions)
Gel doc 2000, by Bio-Rad (3 mentions)
Precast sds polyacrylamide gel, by Bio-Rad (3 mentions)
Ip buffer, by Beyotime (3 mentions)
Gapdh, by Abcam (3 mentions)
Ecl reagent, by Thermo Fisher Scientific (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Ab68155, by Abcam (3 mentions)
Ab189860, by Abcam (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Exoab cd63a 1, by System Biosciences (3 mentions)
Primary antibody dilution buffer, by Beyotime (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg secondary antibody, by MultiSciences Biotech (2 mentions)
87 protocols using protein lysis buffer
1
Protein Isolation and Antibody Validation
2
Western Blot Analysis of AMPD2 Expression
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Forty-eight hours after NovelmiRNA-25 mimic transfection, HEK293T cells and PBMCs from 5 SLE patients and 5 HCs were lysed using protein lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL, USA) at 4 °C for 20 min. Protein samples were separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Antibodies against AMPD2 (ab31537, MULTI SCIENCES, Hangzhou, China) were diluted with primary antibody dilution buffer (Beyotime Institute of Biotechnology) at a 1:400 dilution, while those against GAPDH (GOOD HERE, Hangzhou, China) were diluted 1:1000. The membranes were then washed with TBST buffer five times for 5 min each and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution) (MULTI SCIENCES) for 1.5 h at 37 °C. Bands were detected using enhanced chemiluminescence and visualized with a Gel Doc 2000 (BioRad, Hercules, CA, USA).
3
Investigating NF-κB Signaling Pathway Activation
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Whole‐cell lysates were collected in protein lysis buffer (Beyotime), and the protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime). The protein samples were separated by electrophoresis (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) and then transferred to polyvinylidene difluoride membranes (Millipore). Subsequently, the membranes were incubated with primary and secondary antibodies in TBST containing 5% milk. Primary antibodies against phosphorylation‐p65 (p‐p65, Rabbit, ABclonal), p65 (Rabbit, ABclonal), IkappaB kinase (IKK) α (IKKα, Rabbit, Affinity), phosphorylation‐IKKα/β (p‐IKKα/β, Rabbit, Affinity), and TMUB1 (Rabbit, Abcam) were included in the study. Histone H3 antibody (Mouse, ABGENT) and β‐actin antibody (Mouse, Santa Cruz Biotechnology) were used to normalize the protein expression. Western blots were developed using ECL detection reagents (Beyotime).
4
Western Blot Analysis of Apoptosis Regulators
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Total proteins of transfected AGS, BGC-823, MGC-803 and GES-1 cells were extracted using protein lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) supplemented with protease inhibitor cocktail (Pierce) at 4°C for 30 min. The concentrations of these cell total proteins were determined using the BCA assay kit. Protein samples (20 μg/lane) were separated using 8–12% SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk for 1.5 hr at 37°C, the membranes were then incubated with the primary antibodies at 4°C overnight, the antibody of caspase-3, caspase-9, Bcl-2 (Cell Signaling Technology, Beverly, MA, USA), GAPDH (Santa Cruz, CA, United States) and HSP70 were diluted with TBST in the concentration of 1:1000, the antibody of UL138 was diluted to 1:2000. The membranes were then washed with TBST buffer for 3 × 5 minutes and incubated with the secondary antibodies (HRP-conjugated goat anti-rabbit IgG (Beyotime Institute of Biotechnology, Beijing, China) for 2 hr at room temperature. The bands were detected using enhanced chemiluminescence and visualised by a Gel Doc 2000 (BioRad, USA) and the data was analyzed by Image J.
5
Western Blot Analysis of IGF2BP1 Expression
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Caski cells were lysed in Protein Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer's instructions. Protein concentration was determined using the BCA Protein assay kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. For each well, protein (60 µg) was separated using 10% SDS-PAGE and then transferred to a PVDF membrane (Thermo Fisher Scientific, Inc.). Following 3 h incubation at room temperature in TBST containing 5% nonfat dried milk (Yili Group, Beijing, China), the PVDF membrane was incubated with primary antibodies against IGF2BP1 (cat. no. ab82968) and GAPDH (cat. no. ab9485)(1:100; both from Abcam, Cambridge, MA, USA) at room temperature for a further 3 h and then washed with TBST three times. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. ab97051, 1:5,000; Abcam) for 40 min at room temperature. The membrane was then washed three times with TBST and the immune complexes on the PVDF membrane were detected using the ECL Western Blotting kit (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. ImageJ software v.1.48 (National Institutes of Health, Bethesda, MD, USA) was used to analyze relative protein expression, which was presented as the density ratio versus GAPDH.
6
Protein Extraction and Immunoprecipitation Protocols
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The cells were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. The protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25–35 μg) were separated on a 10% polyacrylamide precast SDS gel (Bio-Rad, Richmond, CA, USA) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). For IP experiments, the cells were lysed in IP buffer (Beyotime, China) and incubated with IP-grade antibodies, followed by pull-down with protein A/G beads (161-4023) (Bio-Rad, Richmond, CA, USA) for subsequent immunoblot analyses.
For pull-down assay, THP-1 cell lysates were collected and centrifuged at 8000 × g. The supernatant was then transferred to another tube and the cell debris was thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h of preincubation with shaking at 4°C to remove unspecific binding proteins, followed by incubation with indicated doses of biotin-GPA for 6 h. Beads were washed with IP buffer for three times and boiled in SDS buffer.
For pull-down assay, THP-1 cell lysates were collected and centrifuged at 8000 × g. The supernatant was then transferred to another tube and the cell debris was thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h of preincubation with shaking at 4°C to remove unspecific binding proteins, followed by incubation with indicated doses of biotin-GPA for 6 h. Beads were washed with IP buffer for three times and boiled in SDS buffer.
7
Protein Extraction and Western Blot Analysis
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Total proteins were extracted with the protein lysis buffer (Beyotime) and quantified using a BCA protein quantification kit (Beyotime). The protein samples were isolated by polyacrylamide gel electrophoresis, and the proteins on the gel were transferred to polyvinylidene fluoride membranes. The membranes were closed with 5% skim milk for 1 h and treated with the corresponding specific primary antibody and horseradish peroxidase-labeled secondary antibody in sequence. Finally, images were obtained using the Ultrasensitive ECL Chemiluminescence Kit (Beyotime), and bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Western Blot Analysis of Vascular Proteins
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Total protein of cells and vascular tissues was extracted with protein lysis buffer (Beyotime, China) and protease inhibitor mix (Thermo Fisher Scientific, USA). A BCA kit (Thermo Fisher Scientific, USA) was used for protein quantification. The protein extract denatured at 98°C was then separated by 10% SDS-PAGE, transferred to a 0.4 μm pore size PVDF membrane and blocked in a 5% skim milk solution for 1 h at room temperature. After washing in TBST solution, the membranes were incubated overnight at 4°C in the following antibody solutions: anti-α-SMA (1:10,000; Proteintech, Cat# 55135-1-AP, China), anti-SMMHC (1:1,000; Proteintech, Cat# 21404-1AP), anti-CNN1 (1:1,000; Cell Signaling Technology, Cat# 17819, USA), anti-PCNA (1:10,000; Abcam, Cat# ab92552, USA), anti-CBX3 (1:1,000; Proteintech, Cat #11650-2-AP), and anti-β-actin (1:1,000; Proteintech, Cat# 66009-1-Ig). On the following day, the membranes were washed and incubated in the corresponding secondary antibody solutions (1:10,000, EasyBio, China, Cat# BE0132-100) for 1 h at room temperature. An ECL Chemiluminescence Kit (Millipore, USA) was used to detect the protein signal. ImageJ was used for gray value analysis.
9
Ischemic Penumbra Tissue Analysis
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Ischaemic penumbra tissues were collected after 24 h of perfusion for western blotting. The brain tissue samples were mechanically homogenized in protein lysis buffer (Beyotime, China). The lysates were centrifuged at 12,000×g for 10 min at 4°C. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime, China). The samples (50 μg per lane) were separated by 10% SDS‐PAGE and electrotransferred onto a nitrocellulose membrane. The non‐specific proteins on the membrane were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against GFAP (1:1000), MMP‐9 (1:300), RGS5 (1:300), laminin (1:300) and PDGFR‐β (1:300). After washing three times for 5 min, the membrane was incubated with secondary antibodies (1:5000) for 1 h at 37°C. The bands were visualized by incubation with the chemiluminescence reagents provided in the ECL kit for 1 min. Finally, the protein bands were imaged, and the grey value of each band was analysed using a chemiluminescence imaging system (Bio‐Rad, Hercules, CA, USA). The grey value of the target band relative to that of the β‐actin band was calculated, and then, the value was normalized to that of the sham group.
10
Western Blot Analysis of Myocardial Proteins
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Total protein from myocardial tissues or cells was extracted using Protein Lysis Buffer (Beyotime, Shanghai, China). The protein concentration was determined using BCA Protein Assay Kit (Beyotime). Equal amounts of protein were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with defatted milk and incubated with primary antibodies overnight at 4°C. After washing with PBS, the membranes were incubated with secondary antibodies goat antirabbit IgG (1 : 10,000, ab6721) for 2 h at room temperature. Finally, the protein bands were visualized with enhanced chemiluminescence detection kits (Amersham Pharmacia Biotech, UK) and analyzed by ImageJ software. The primary antibodies against Bax (1 : 1000, ab32503), collagen I (1 : 1000, ab34710), SH2B3 (1 : 1000, ab191904), collagen III (1 : 1000, ab7778), Bcl-2 (1 : 1000, ab182858), fibronectin (1 : 1000, ab2413), and GAPDH (ab245356) were purchased from Abcam (Cambridge, USA) and GAPDH acted as a loading control. This assay was performed for 3 times (n = 3).
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