RF/6A cells in each group were lysed using RIPA buffer (Beyotime Institute of Biotechnology). Nucleus proteins of RF/6A cells of each group were also obtained through using nucleus protein extraction kit (Beyotime Institute of Biotechnology). Cell lysate (30 µg protein per lane) was then separated by 12% SDS-PAGE. The separated proteins were transferred onto nitrocellulose membranes (Beyotime Institute of Biotechnology), which were then blocked with Tris-buffered saline containing Tween-20 with 5% non-fat milk. The membrane was incubated overnight with primary antibodies to GAPDH (1:1,000), VEGF (1:1,000) and VEGFR-2 (1:1,000), and then incubated with a horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology; cat. no. P0239; 1:1,000) for 1 h at room temperature. The labeled bands were visualized and quantified using a chemiluminescence imaging system (Tanon 5000; CliNX). CliNX analysis software (version 1.7.0) was used to scan the gels and determine the gray value. The ratio of the target protein gray value to that of β-actin represented the relative expression levels of the target protein.
Nitrocellulose membrane
Manufactured by Beyotime
Sourced in China, United States
Nitrocellulose membranes are a type of porous membrane made from nitrocellulose. They are commonly used in various laboratory applications for the transfer and immobilization of proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (15 mentions)
Bca protein assay kit, by Beyotime (14 mentions)
Odyssey infrared scanning system, by LI COR (7 mentions)
Cell lysis buffer, by Beyotime (6 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Exosome precipitation kit, by System Biosciences (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
X omat bt film, by Beyotime (3 mentions)
L ascorbic acid, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
β glycerol phosphate, by Merck Group (3 mentions)
Anti β actin, by ABclonal (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (3 mentions)
Lysis buffer, by Beyotime (3 mentions)
Anti pcna, by Proteintech (3 mentions)
Protein assay kit, by Beyotime (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
70 protocols using nitrocellulose membrane
1
Quantitative Western Blot Analysis
2
Protein Quantification and Western Blot Analysis
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Total protein was obtained from the cells with RIPA lysis buffer (Beyotime) following the manufacturer’s protocol. The concentration of protein was measured using BCA Protein Assay Kit (Beyotime), and then equal amounts of protein samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and finally transferred onto nitrocellulose membranes (Beyotime). The membranes were then blocked with BSA for 30 mins and incubated with primary antibodies at 4 °C overnight, followed by the secondary antibody for 1 h. Immunoreactive protein bands were detected using an Odyssey scanning system (SYSTEM/Manufacturer Info). The protein expression levels of the kinases were normalized against β-actin.
3
Protein Expression Analysis in Cells
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Tissue samples and cultured cells were lysed using the RNA immunoprecipitation assay (RIPA) buffer mixed with protease inhibitors (Sigma‐Aldrich), followed by quantitation with the BCA protein assay kit (Applygen). Protein (40 µg per lane) was separated on sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by being transferred to nitrocellulose membranes (Beyotime). After blocking with 5% nonfat‐dried milk, membranes were then incubated using the primary antibodies against light Chain 3 (LC3)I/II (1:3000, ab51520; Abcam), p62 (1:1000, ab56416; Abcam), cyclinD1 (1:2000, ab226977; Abcam), cleaved‐caspase‐3 (1:500, ab49822; Abcam), or β‐actin (1:2500, ab8227; Abcam) for 12 h at 4°C. After being washed, the secondary antibody (1:4000, ab205718; Abcam) was used to incubate the membranes for 2 h. Protein bands were visualized by an enhanced chemiluminescent kit (Applygen). Protein expression was normalized to β‐actin and quantified using ImageJ software.
4
Western Blot Analysis of 6×His-tagged Proteins
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The samples were mixed with 5× SDS-PAGE sample loading buffer and boiled for 5 min. Protein bands were separated by SDS-PAGE with a 12% separating gel and a 5% stacking gel, and transferred to nitrocellulose membranes (Beyotime, Shanghai, China). The membranes were incubated with 5% nonfat dried milk in TBS with 0.1% Tween-20 (TBST) for blocking, and then incubated with 6 × His-tag mouse monoclonal antibody (1:2500 dilution, EnoGene Biotech, Nanjing, China). After washing with TBST, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 dilution, EnoGene Biotech, Nanjing, China). Finally, the immunoreactive proteins were detected with BeyoECL Star (Beyotime, Shanghai, China).
5
Quantification of Protein Expression by Western Blot
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Total proteins were extracted by RIPA lysis buffer (Beyotime, China) together with PMSF (Beyotime, China). The concentration of total protein was determined by BCA protein assay kit (Beyotime, China). After being separated by 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, proteins lysates were transferred to nitrocellulose membranes (Beyotime, China). The membranes were blocked with 5% non-fatty milk for 1 h at room temperature and then immunoblotted at 4 °C overnight with primary antibodies: anti-WBP2 (1:1000, Proteintech, USA), anti-AKT (1:1000, Proteintech, USA), anti-p-AKT (1:1000, Abcam, USA) and anti-β-actin (1:10,000, Abclonal, China). After incubated with diluted secondary antibodies for 1 h at room temperature, the bands were scanned and analyzed by Odyssey Infrared scanning system (LI-COR Biosciences, USA). Original western blots were shown in Fig. S1 .
6
Western Blot Analysis of Autophagy Markers
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Liver tissues were cut into pieces and lysed with RIPA buffer (Beijing Solarbio Science
& Technology Co., Ltd., Beijing, China) supplemented with protease inhibitors (Beijing
Applygen Co., Ltd., Beijing, China) on ice for 30 min and centrifuged at 12,000 × g for 20
min at 4°C. The protein concentration was determined by the BCA method according to the
manufacturer’s instructions (Beyotime Institute of Biotechnology, Haimen, China). Total
proteins (20 μg) were separated via 12–15% SDS polyacrylamide gel electrophoresis (PAGE)
and transferred to nitrocellulose membranes (Beyotime Institute of Biotechnology). After
blocking at room temperature for 2 h with 5% non-fat milk in TBS with 0.1% Tween 20, the
membranes were incubated overnight at 4°C with antibodies against BECN1 (cat. no. 3495),
Atg5 (cat. no. 12994), Atg7 (cat. no. 8558), and Akt (cat. no. 4691), p-Akt (Thr308) (cat.
no. 13038), Raptor (cat. no. 2280), P-Raptor (Ser792) (cat. no. 2083), AMPKα (cat. no.
5832), P-AMPKα (Thr172) (cat. no. 2535), ULK1(cat. no. 8054), P-ULK1 (Ser555) (cat. no.
5869), β-actin (cat. no. 4970) and HRP-conjugated secondary antibodies (cat. no. 7074) at
room temperature for 1.5 h; all antibodies were purchased from Cell Signaling Technology
(Danvers, MA, USA). Signals were visualized with Amersham ECL substrates, and the relative
levels of protein in each group were normalized to β-actin.
& Technology Co., Ltd., Beijing, China) supplemented with protease inhibitors (Beijing
Applygen Co., Ltd., Beijing, China) on ice for 30 min and centrifuged at 12,000 × g for 20
min at 4°C. The protein concentration was determined by the BCA method according to the
manufacturer’s instructions (Beyotime Institute of Biotechnology, Haimen, China). Total
proteins (20 μg) were separated via 12–15% SDS polyacrylamide gel electrophoresis (PAGE)
and transferred to nitrocellulose membranes (Beyotime Institute of Biotechnology). After
blocking at room temperature for 2 h with 5% non-fat milk in TBS with 0.1% Tween 20, the
membranes were incubated overnight at 4°C with antibodies against BECN1 (cat. no. 3495),
Atg5 (cat. no. 12994), Atg7 (cat. no. 8558), and Akt (cat. no. 4691), p-Akt (Thr308) (cat.
no. 13038), Raptor (cat. no. 2280), P-Raptor (Ser792) (cat. no. 2083), AMPKα (cat. no.
5832), P-AMPKα (Thr172) (cat. no. 2535), ULK1(cat. no. 8054), P-ULK1 (Ser555) (cat. no.
5869), β-actin (cat. no. 4970) and HRP-conjugated secondary antibodies (cat. no. 7074) at
room temperature for 1.5 h; all antibodies were purchased from Cell Signaling Technology
(Danvers, MA, USA). Signals were visualized with Amersham ECL substrates, and the relative
levels of protein in each group were normalized to β-actin.
7
Western Blot Analysis of Liver Proteins
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The liver tissues were stored at −80°C. Tissue samples were homogenized in ice-cold RIPA lysis buffer (Millipore, Billerica, MA, USA) for protein extraction. Tissue debris was removed by centrifugation, and the resulting supernatants were collected and analyzed for protein concentration by the BCA protein assay kit. The protein was separated on a 10% SDS polyacrylamide gel and then transferred to nitrocellulose membranes (Beyotime Biotech, Shanghai, China). The membranes were incubated with specific primary antibodies overnight at 4°C. The primary antibodies included anti-mouse β-actin (Beyotime Biotech, Shanghai, China), anti-rabbit ATF6, anti-rabbit CHOP, anti-rabbit Wnt3a, and anti-rabbit β-catenin (Company ABclonal, Inc., Wu Han, China). After washing, the membranes were allowed to react with diluted horseradish peroxidase-conjugated secondary antibodies, including goat anti-rabbit IgG antibody and goat anti-mouse IgG (Beyotime Biotech, Shanghai, China) at room temperature for 2 h. An enhanced chemiluminescence system (Beyotime Biotech, Shanghai, China) was used to visualize antibody-antigen complexes.
8
Western Blot Analysis of PTEN in MSCs and CM
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Briefly, the MSCs were washed with PBS three times and collected using cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Cell lysates were incubated on ice for 30 min. The protein concentration was determined using bicinchoninic acid protein assay reagents (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Equal amounts of protein (50 µg/sample) were loaded on each lane and separated by electrophoresis in 12% sodium dodecyl sulfate polyacrylamide gel and electrotransferred to nitrocellulose membranes (Beyotime Institute of Biotechnology). The membrane was incubated in blocking buffer for 1 h at RT and then incubated overnight at 4°C with monoclonal rabbit anti-mouse PTEN primary antibodies (cat. no. 217702; dilution, 1:1,000; R&D Systems, Inc., Minneapolis, MN, USA). The blots were rinsed with Tris-buffered saline and Tween-20 (EMD Millipore, Billerica, MA, USA) three times, incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (cat. no. A0216; dilution, 1:1,000; Beyotime Institute of Biotechnology) for 60 min and detected by chemiluminescence using ECL Hyperfilm (EMD Millipore). The CM samples were filtered through a 0.22 µm membrane (EMD Millipore) and equally concentrated using a 10,000 molecular weight cut-off (catalog no., 42406; EMD Millipore). Then, PTEN was detected in the CM.
9
Protein Extraction and Western Blotting
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At the scheduled time points after incubation, cellular proteins in samples were extracted by lysing with radioimmunoprecipitation assay (RIPA; Beyotime) buffer. Proteins (20 μg) were resolved by SDS–PAGE (Beyotime) and transferred to nitrocellulose membranes (Beyotime). After closure with 5% skim milk or 5% BSA for at least 1 h, membranes were incubated separately with primary antibodies against β-Actin (1:1000), NFATc1 (1:1000), MMP9 (1:1000), CTSK (1:1000), IκB-α (1:1000), phospho-P38 (1:1000), P38 (1:2500), phospho-JNK (1:5000), JNK (1:2000), phospho-ERK (1:1000), ERK (1:1000) and TRAF6 (1:5000) at 4 °C overnight. The corresponding secondary antibody dilutions (1:1000) were then added for 1 h at room temperature after washing membranes adequately with Tris-buffered saline Tween (TBST) for an appropriate time and frequency. Finally, to show the protein bands, membranes were treated with enhanced chemiluminescence (ECL; Yeasen) reagents and imaged using Image Lab 3.0. Protein quantification (relative grey level of the bands) was measured using ImageJ software.
10
Quantifying Genomic 5-Hydroxymethylcytosine
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Genomic DNA was extracted from BM-MSCs using a TIANamp Genomic DNA Kit (Tiangen, China) according to the manufacturer’s instructions. A NanoDrop One system (Thermo Fisher Scientific, USA) was used to quantify the DNA concentration. DNA extracts were stored at − 80 °C until use. DNA samples were loaded on nitrocellulose membranes (Beyotime, China). After being incubated at 60 °C for 1 h and blocked with 5% nonfat milk for 30 min at room temperature, the membrane was incubated with an anti-5hmC antibody (0.2 µg/ml, Abcam) at 4 °C overnight. The blots were visualized using enhanced chemiluminescence reagents (Beyotime, China) on a Tanon 5200 image analyser (Tanon, China). To ensure equal loading, the membrane was stained with methylene blue (Aladdin, China) after immunoblotting. The density of the dots was analysed with ImageJ software (National Institutes of Health, version 1.51k).
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