Protein extraction from the aortic tissues was performed using tissue lysis buffer (Solarbio, Beijing, China) containing 1% phenylmethanesulfonyl fluoride and protein was quantified using a BCA protein assay kit. The protein (25 µg) was separated on a 10% SDS-PAGE gel (Beijing Solarbio Science & Technology Co., Ltd.) and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). Subsequently, the membrane was blocked with 5% nonfat dried milk for 2 h at room temperature and then incubated with rabbit primary antibodies against NF-κB, TNF-α and polyclonal rabbit anti-rat β-actin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4967), GLUT4 and IRS1 (1:2,000; Abcam) and polyclonal rabbit anti-rat PI3K (1:1,000, Sigma-Aldrich; cat. no. 5295) overnight at 4°C. The membrane was washed with 0.1% PBST three times for 5 min. Subsequently, the membrane was incubated with HRP-labeled goat anti-rabbit IgG (cat. no. GGHL-15P; ICL, Inc., Portland, OR, USA) secondary antibodies for 1 h at room temperature. The same membranes probed for β-actin served as an internal standard. The relative density of protein to β-actin was analyzed using ImageJ software (version 1.43; National Institutes of Health, Bethesda, MA, USA).
Sds page gel
Manufactured by Solarbio
Sourced in China, United States
SDS-PAGE gel is a type of gel electrophoresis used to separate proteins based on their molecular weight. It allows for the separation and analysis of complex protein mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Solarbio (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Horseradish peroxidase labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Quantiscan software, by Biosoft (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by Proteintech (2 mentions)
Tissue lysis buffer, by Solarbio (1 mentions)
Ecl chemiluminescence solution, by Beyotime (1 mentions)
Skim milk, by Solarbio (1 mentions)
Lysis buffer, by Solarbio (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab2539, by Abcam (1 mentions)
Ab190684, by Abcam (1 mentions)
Phospho tau, by Cell Signaling Technology (1 mentions)
16 protocols using sds page gel
1
Aortic Tissue Protein Extraction and Analysis
2
Protein Extraction and Western Blot Analysis
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Protein extraction
was conducted using a RIPA lysis buffer (Beyotime, China) along with
the addition of both phosphatase and protease inhibitors. Following
protein extraction, precise protein concentration measurements were
carried out by utilizing a BCA protein quantification kit. Subsequently,
protein samples were combined with a 10% SDS-PAGE gel (Solarbio, China)
to effect protein separation through gel electrophoresis. Upon completion
of the separation process, proteins were transferred onto poly(vinylidene
fluoride) (PVDF) membranes to facilitate subsequent immunodetection.
To mitigate nonspecific binding, PVDF membranes were subjected to
a blocking procedure involving 5% skim milk. During the immunodetection
phase, specific antibodies were incubated with the target protein
on the PVDF membrane, a process carried out overnight at 4 °C.
Afterward, suitable secondary antibodies were used to enhance the
signal following the primary antibody reaction. Visualization of protein
bands was achieved through the utilization of an ECL chemiluminescence
solution (Beyotime), enabling reliable visualization of protein bands.
Ultimately, the quantitative analysis of protein bands was performed
using ImageJ software.
was conducted using a RIPA lysis buffer (Beyotime, China) along with
the addition of both phosphatase and protease inhibitors. Following
protein extraction, precise protein concentration measurements were
carried out by utilizing a BCA protein quantification kit. Subsequently,
protein samples were combined with a 10% SDS-PAGE gel (Solarbio, China)
to effect protein separation through gel electrophoresis. Upon completion
of the separation process, proteins were transferred onto poly(vinylidene
fluoride) (PVDF) membranes to facilitate subsequent immunodetection.
To mitigate nonspecific binding, PVDF membranes were subjected to
a blocking procedure involving 5% skim milk. During the immunodetection
phase, specific antibodies were incubated with the target protein
on the PVDF membrane, a process carried out overnight at 4 °C.
Afterward, suitable secondary antibodies were used to enhance the
signal following the primary antibody reaction. Visualization of protein
bands was achieved through the utilization of an ECL chemiluminescence
solution (Beyotime), enabling reliable visualization of protein bands.
Ultimately, the quantitative analysis of protein bands was performed
using ImageJ software.
3
Hippocampal Protein Expression Analysis
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Dissected hippocampal tissues or cell samples were added to lysis buffer (Solarbio, Beijing, China), then ultrasonicated and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and protein quantification was conducted via the Lowery method. Fifty micrograms of total proteins were added to a 10% SDS-PAGE gel (Solarbio, Beijing, China), separated by electrophoresis, and transferred to polyvinylidene fluoride membrane, which was blocked with 5% skim milk (Solarbio, Beijing, China). Membranes were incubated with primary antibodies SORLA (ab190684; Abcam, Cambridge, UK), Aβ (ab2539; Abcam, Cambridge, UK), Phospho-Tau (#20194; Cell Signaling Technology, USA), and GAPDH (#5174; Cell Signaling Technology, USA) were incubated overnight at 4° C. Secondary antibody (#7074; Cell Signaling Technology, USA) were added, and the ECL method was used to measure luminescence. X-ray scanning was used for imaging, and Seion-Image software was used for quantitative analysis of the results.
4
Quantification of Apoptosis Pathway Proteins
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After extracted total protein from NP cells and NP tissues with RIPA buffer (Cell Signaling Technology, USA), the samples were transferred into a polyvinylidene fluoride membrane (PVDF, Millipore), and electrophoresis was conducted to separate the samples with a 10% SDS-PAGE gel (Solarbio, China). The protein concentration was quantified by a BCA assay kit (Sigma-Aldrich; USA). The primary antibodies were TRAIL (Rabbit; 3219; 1 : 1000), DR5 (Rabbit; 8074; 1 : 1000), FADD (Rabbit; 2782; 1 : 1000), caspase8 (Mouse; 9746; 1 : 1000), cleaved caspase8 (Rabbit; 8592; 1 : 1000), caspase3 (Rabbit; 9662; 1 : 1000), cleaved caspase3 (Rabbit; 9661; 1 : 1000), PARP (Rabbit; 9532; 1 : 1000), cleaved PRAP (Rabbit; 5625; 1 : 1000), and GAPDH (Rabbit; 5174; 1 : 1000) specific. All antibodies were bought from Cell Signaling Technology, the USA. Then, we added the secondary antibodies (goat anti-mouse/rabbit IgG; Abcam, UK), which were conjugated with horseradish peroxidase (HRP) (CST, USA). ImageJ software (version 5.0; Bio-Rad, the USA) was applied to analyze and quantify the gray value of the strips. GAPDH was used as a control.
5
Western Blot Protein Detection Protocol
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Protein samples mixed with 5 × SDS-PAGE Loading Buffer were resolved by electrophoresis in a 10% SDS-PAGE gel (Solarbio®,Beijing, China). Proteins were transferred onto a PVDF membrane in transfer buffer (39 mM glycine, 48 mM Tris, 0.037% (w/v) SDS, 20% (v/v) methanol) using a wet procedure at 300 mA for 1 h. The PVDF membrane was blocked with blocking buffer (5% (w/v) non-fat milk dissolved in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% (v/v) Tween 20)) at room temperature for at least 1 h and then washed with TBST buffer 3 times and incubated with His-Tag Mouse Monoclonal antibody (1:10,000, Proteintech, Cat# 66005-1-Ig) for 1.5 h at room temperature. The membrane was washed for 10 min with TBST buffer on an orbital shaker. This was repeated for two additional washes and then the blot was incubated with Goat anti-mouse secondary antibody (1:5000, CWBIO, Cat# CW0102S) at room temperature for 1 h, and washed with TBST as described above. Protein expression was detected with ECL Western HRP substrate (Advansta) on a Fluorescent gel imaging system (MicroChemi, DNR).
6
Protein Extraction, Quantification, and Western Blot Analysis
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The extraction of total protein was finished through the usage of RIPA (CWBio, Beijing, China) and the concentrations of proteins were determined through the usage of BCA Protein Quantification Kit (Vazyme). Next, the proteins were loaded on SDS-PAGE gel (Solarbio) and blotted onto PVDF membranes (Amersham Biosciences, Chicago, IL, USA). After blocking in 5% defatted milk for 1 h, the membranes were cultivated overnight with primary antibodies at 4°C followed by incubation with secondary antibody (ab205719; Abcam, Cambridge, MA, USA) for 1 h at indoor temperature. The blots were examined with an ECL detection kit (Pierce Biotechnology, Rockford, IL, USA). The primary antibodies were: Bcl-2 (ab182858; Abcam), Bax (ab32503; Abcam), cleaved-caspase-3 (c-caspase-3; ab49822; Abcam), PRKACB (ab26322; Abcam) and GAPDH (ab9485; Abcam).
7
Cardiac Protein Extraction and Western Blot Analysis
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A phosphatase inhibitor (Solarbio) was used to extract proteins from cardiac tissue using RIPA lysis buffer (Solarbio, Beijing, China). The BCA protein assay (Thermo Fisher Scientific, American) was used to determine soluble protein concentrations. SDS-PAGE gel (Solarbio) was used to separate 20 μg proteins. Polyvinylidene difluoride membranes (Millipore, American) contained the protein bands. The western blots were cut prior to hybridisation with the primary antibody during the imprinting process. The primary antibody, IDO (sc-53978, Santa Cruz, American), was incubated overnight at 4°C after being blocked for 4 h with blocking buffer (5% skimmed milk, 150 mM NaCl, 20 mM Tris HCl, 0.05% Tween-20, and pH 7.6). Incubate the secondary antibodies (ABCAM), labeled with horseradish peroxidase, for 2 h at room temperature. Image J was used to analyze the densitometric differences between the bands, when they were visualized by chemiluminescence (ECL, Millipore).
8
Western Blot Analysis of NF-κB-p65
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Proteins were extracted from endometrium samples with RIPA buffer containing proteinase inhibitors. The protein concentrations were quantified using BCA Protein Assay Kit (Beyotime, Shanghai, China) and separated in SDS-PAGE gel (Solarbio, Beijing), and then they were transferred onto the PVDF membrane. The membranes were incubated with anti-NF-κB-p65 and anti-GADPH for overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. The blots were visualized using ECL chemiluminescence kit (enhanced), and the band intensity was quantified with ImageJ software.
9
Protein Extraction and Western Blot Analysis
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The total proteins extracted from the tissue were obtained using RIPA buffer (Beyotime, Shanghai, China), which contained PMSF (Beyotime) to inhibit protease degradation. Next, the samples were separated via electrophoresis on an SDS-PAGE gel (Solarbio) and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After that, the membranes were incubated overnight at 4 °C with one of the primary antibodies: γH2AX (BIOSS, Beijing, China) at a 1:500 dilution, CASPASE-3 (HUABIO, Hangzhou, China) at a 1:500 dilution, and β-actin (HUABIO) at a 1:5000 dilution. Next, a horseradish peroxidase-labeled secondary antibody (Thermo Fisher, Waltham, MA USA) was added and incubated at room temperature for 1 h. Finally, the protein bands were developed using an ECL Western Blot Kit (Millipore, Bedford, MA, USA), and the relative intensities of the bands were determined using Quantiscan software (Biosoft, Cambridge, United Kingdom).
10
Western Blot Analysis of Signaling Proteins
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The total proteins extracted from the tissue were obtained using RIPA buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China), which contained PMSF (Beyotime Biotechnology Co., Ltd.) to inhibit protease degradation. Next, the samples were separated via electrophoresis on an SDS-PAGE gel (Solarbio Biotechnology) and then transferred onto polyvinylidene fluoride membranes (Millipore, Boston, Massachusetts, USA). Afterward, the membranes were incubated with one of the primary antibodies (IL-11Rα, 1:500 dilution, HuaBio Biotechnology Co., Ltd.; GP130, 1:500 dilution, ABclonal, Wuhan, China; NSUN5, 1:500 dilution, Thermo Fisher, Waltham, Massachusetts, USA; JAK2, 1:1000 dilution, ABclonal; P-JAK2, 1:500 dilution, ABclonal; STAT3, 1:1000 dilution, ABclonal; P-STAT3, 1:500 dilution, ABclonal; CD3, 1:1000 dilution, HuaBio Biotechnology Co., Ltd.; and β-actin, 1:5000 dilution, HuaBio Biotechnology Co., Ltd.) overnight at 4°C. Next, a horseradish peroxidase-labeled secondary antibody (Thermo Fisher) was added and incubated for 1 h at room temperature. Lastly, the protein bands were developed using a chemiluminescence detection kit (Millipore, Bedford, MA, USA), and the relative intensities of the bands were determined using Quantiscan software (Biosoft, Cambridge, United Kingdom).
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