Hippocampal tissues were mixed and homogenized with sample buffer (pH 7.6; 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) on ice and boiled for 10 minutes. Thirty micrograms of protein was heated at 95°C for 5 minutes and then cooled on ice for 5 minutes. The protein was fractionated on a 4–12% SDS-PAGE Bis-Tris gel using an SDS-PAGE system (Bio-Rad, CA, USA). The proteins were transferred onto nitrocellulose membranes (Whatman, Kent, UK). Following blocking in 5% nonfat milk in 0.01% Tween PBS (PBST), the membranes were incubated with optimally diluted primary antibodies overnight at 4°C. Following washing with PBST, the membranes were incubated with the appropriate anti-mouse or anti-rabbit secondary antibody diluted 1:10,000 for 1 h at room temperature and visualized using the Odyssey Infrared Imaging System. The protein bands were quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA).
Sds page system
Manufactured by Bio-Rad
Sourced in United States
The SDS-PAGE system is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It utilizes sodium dodecyl sulfate (SDS) and polyacrylamide gel electrophoresis (PAGE) to denature and separate proteins, respectively. The core function of the SDS-PAGE system is to provide a standardized and reproducible method for protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Digital science 1d software, by Kodak (1 mentions)
Chemiluminescence, by Beyotime (1 mentions)
Ab214430, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab216494, by Abcam (1 mentions)
Electrotransfer, by Bio-Rad (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemiluminescence detection system, by GE Healthcare (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
Cell lytic buffer, by Merck Group (1 mentions)
Hdac3, by Cell Signaling Technology (1 mentions)
12 protocols using sds page system
1
Protein Expression Analysis in Hippocampus
2
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted by RIPA buffer (Sigma-Aldrich) containing 1% protease inhibitor (cocktail, CalbioChem, Darmstadt, Germany), and the concentration of proteins was determined by using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins (70 μg proteins per sample) were denatured at 95°C for 5 min and then separated by SDS-PAGE system (Bio-rad). After transferring the electrophoretic protein onto PVDF membranes (Millipore, Billerica, MA, USA) by electrotransfer (Bio-rad) and blocking with 5% non-fat milk at room temperature for 90 min, the proteins were probed with primary antibodies against Bcl-2 (ab196495), Bax (ab216494), cleaved caspase3 (c-cas3, ab214430), caspase3 (cas3, ab13847), HMGB1 (ab18256), and GAPDH (ab181602) (Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with the appropriate peroxidase-conjugated secondary antibodies at room temperature for 1 h. Blots were developed by chemiluminescence (Beyotime), and then an X-ray film was scanned. The gray ratios of the target protein bands were accurately determined by an Image-J analysis system (NIH, Rockville Pike, Bethesda, MD, USA).
3
Western Blot Analysis of Signaling Pathways
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Vehicle‐ or compound‐treated cells were lysed 48 h after drug incubation with Cell Lytic buffer (Sigma; 150 mm NaCl, 0.41% bicine, 2% EDTA) supplemented with complete protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche). Protein concentration was quantified with BCA assay, and normalized samples were resolved with Bio‐Rad SDS/PAGE system on 8–12% protein gels. Signal detection was performed with chemiluminescence detection system (GE Healthcare, Chicago, IL, USA).
Poly(ADP‐ribose) polymerase (PARP), caspase 3, p‐ERK1/2 (Thr202/Tyr204), total ERK1/2, p‐p38 (Thr180/Tyr182), total p38, p‐BRAF (Ser445), p‐MEK1/2 (Ser217/221), total MEK1/2, SOS1, acetyl‐H3 (Lys9/14), p‐STAT3 (Ser727), total STAT3, UBE2C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, Sirt1, β‐actin, and HRP‐conjugated anti‐rabbit were obtained from Cell Signaling (Danvers, MA, USA); SOS2 was purchased from Abcam (Cambridge, UK); FBXO3 was purchased from Sigma Aldrich (St. Louis, MO, USA); FBXW10 was purchased from Novus Biologicals (Littleton, CO, USA).
Poly(ADP‐ribose) polymerase (PARP), caspase 3, p‐ERK1/2 (Thr202/Tyr204), total ERK1/2, p‐p38 (Thr180/Tyr182), total p38, p‐BRAF (Ser445), p‐MEK1/2 (Ser217/221), total MEK1/2, SOS1, acetyl‐H3 (Lys9/14), p‐STAT3 (Ser727), total STAT3, UBE2C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, Sirt1, β‐actin, and HRP‐conjugated anti‐rabbit were obtained from Cell Signaling (Danvers, MA, USA); SOS2 was purchased from Abcam (Cambridge, UK); FBXO3 was purchased from Sigma Aldrich (St. Louis, MO, USA); FBXW10 was purchased from Novus Biologicals (Littleton, CO, USA).
4
Western Blot Analysis of Protein Markers
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Western blot (WB) was carried out using the SDS/PAGE system (Bio‐Rad, Philadelphia, PA, USA), including the precast gradient gel 4–20% and TurboTransfer system, according to the manufacturer’s instructions. The intensities of bands were detected using genetools software (SynGene, Frederick, MD, USA) and standardized by the intensity of α‐tubulin or β‐actin (both antibodies were from Sigma). All other antibodies used were purchased form Cell Signaling Technology (Danvers, MA, USA), with the exception of caspase‐2 (EMD Millipore, Burlington, MA, USA), phospho‐CDC25A(Ser76) (Abcam, Cambridge, MA, USA) and phospho‐CDK2(Tyr15) (Novus, Centennial, CO, USA).
5
Epithelial-Mesenchymal Transition Signaling
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E-cadherin antibody was purchased from BD Biosciences. Plakoglobin and Paxillin (PXN) antibodies were purchased from Millipore. Antibodies against Snail1, mitogen-activated protein kinase (ERK1/2), phospho-p44/42 ERK1/2, SRC, phospho-SRC (pY416), focal adhesion kinase (FAK), phospho-PXN (pY118), phospho-Akt (pS473), β-actin and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology. Phospho-FAK (pY861) antibody was purchased from Thermo Scientific.
Cells are grown on 6-well plates and treated with compounds or DMSO as control for 48 h. Cells were rinsed with PBS and lysed with RIPA buffer supplemented with protease / phosphatase inhibitor cocktail (Promega). Protein concentration was quantified and equalized protein loads were resolved with Bio-Rad SDS-PAGE system using 8% to 12% polyacrylamide gels and transferred to PVDF membranes. Immunoblotting were performed with the antibodies listed above and bound antibodies were detected by chemiluminescence using Amersham ECL Prime Western Blotting reagent (GE Healthcare).
Cells are grown on 6-well plates and treated with compounds or DMSO as control for 48 h. Cells were rinsed with PBS and lysed with RIPA buffer supplemented with protease / phosphatase inhibitor cocktail (Promega). Protein concentration was quantified and equalized protein loads were resolved with Bio-Rad SDS-PAGE system using 8% to 12% polyacrylamide gels and transferred to PVDF membranes. Immunoblotting were performed with the antibodies listed above and bound antibodies were detected by chemiluminescence using Amersham ECL Prime Western Blotting reagent (GE Healthcare).
6
Quantitative Real-Time PCR and Western Blot Analysis
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Extracted RNA was reverse transcribed in a thermal cycler using RT enzyme. The real-time PCR was carried out in ViiA 7 system using TaqMan® gene expression assays. The cycling conditions were 50 °C for 2 min and 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The comparative ΔΔCt method was applied to calculate fold change. For endogenous controls, GAPDH was used for Ahr, CYP1A1 and SOX4, and RNU6B was used for miR-212/132. System, reagents and kits for real-time PCR were all from Applied Biosystems, Grand Island, NY. For western blot, cell were lysed by RIPA lysis buffer system (Santa Cruz Biotechnology), lysate was then fractionated using SDS-PAGE system (Bio-Rad, Richmond, CA). The Ahr, CYP1A1, SOX4 and β-actin were detected using their rabbit polyclonal antibodies (Santa Cruz Biotechnology). The intensities of the protein bands were quantified via software [http://imagej.nih.gov/ij/download.html ] ImageJ v.1.48 [52 (link)].
7
Quantifying Protein Levels via Western Blot
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Protein levels were measured using Western blot analysis [49 (link)]. For whole lung homogenates, the lung tissue was homogenized in RIPA buffer (Thermo Fisher; catalog #89900) and supplemented with protease (Roche; reference #11836170001) and the phosphatase inhibitor sodium orthovanadate (New England Biolabs (NEB), Ipswich, MA, USA; item #P0758) and sodium fluoride (NEB; item #P0759). For cell culture experiments, cellular lysates from were collected in RIPA buffer supplemented with protease and phosphatase inhibitors. Western blotting was performed using an SDS-PAGE system (Bio-Rad, Hercules, CA, USA; item #8658004). Samples were electrophoresed in a 4-20% Tris gel (Bio-Rad; item #4568095) in Tris running buffer, transferred to a PVDF membrane (Bio-Rad; item #1704157), and probed with anti-EGFR (p- and total), anti- ERK1/2 (p- and total), anti-JNK1/2 (p- and total), anti-p38 (p- and total), anti-AKT (p- and total-), anti-HIF1α, anti-PARP (cleaved- and total-), and anti-caspase 3 (cleaved- and total-) primary antibodies (Cell Signaling Technologies, Danvers, MA, USA). Protein band densitometry analysis was completed using ImageJ software.
8
SDS-PAGE Protein Separation and Analysis
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Protein extracts were separated by SDS-PAGE gels following Laemmli procedure [20 (link)]. Essentially, 25 µg of protein from each sample was diluted to the same concentration and mixed with Laemmli buffer, incubated for 5 min at 95 °C and loaded into hand-cast 12% Tris-HCl gels. The proteins were separated under reducing and denaturing conditions using the Bio-Rad SDS-PAGE system, for 20 min applying a 120 V voltage, and then for 30 min applying a 200 V voltage. The gels were then incubated in a fixation solution (40% methanol, 10% acetic acid) for 30 min, stained with 0.12% Colloidal Coomassie Blue G250 in 20% methanol overnight, and destained with 20% methanol until an optimal contrast was achieved. Gels were scanned with ChemiDoc system and analyzed with ImageLab (version 6.1, BioRad Laboratories, Hercules, CA, USA) for automatic detection of protein bands.
9
Quantitative Protein Analysis of Cas9
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Cells were lysed and protein was extracted from cultured cells using RIPA buffer (Thermo Fisher) and the protein concentration was quantified using BCA Protein Assay Kit (Thermo Fisher). SDS-PAGE system (Biorad) was next used to electrophorese approximately 20 µg of protein per sample. Separated proteins were transferred to solid-phase membrane supports using the BIO-RAD blotting system and the primary antibodies (anti-Cas9 from Cell Signaling and anti-GAPDH from ProteinTech, Rosemont, IL, USA) were used to detect protein expression levels.
10
Western Blot Analysis of TNF-α Signaling
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Protein extracted from mice by RIPA lysis buffer (Solarbio, China) was measured by BCA kit (Abcam, UK) to determine the protein concentration, followed by the separation of SDS-PAGE system (Bio-Rad, USA) and subsequently transferred to PVDF membrane (Millipore, Germany). Then, membrane was blocked by blocking buffer (Beyotime, China) at 4°C for 4 h, followed by the incubation with the primary antibodies of TNFα (ab183218, 1:1000), TNFR1 (Abcam, ab223352, 1:1000), TNFRSF1A Associated Via Death Domain (TRADD) (Abcam, ab110644, 1:1000), RIP (Abcam, ab202985, 1:1000), TNF Receptor Associated Factor 2 (TRAF2) (Abcam, ab244317, 0.4 μg/mL), p-Inhibitor of Nuclear Factor Kappa B Kinase Subunit Beta (p-IKKβ) (Cell Signaling, #2697, 1:1000), IKKβ (Cell Signaling, #8943, 1:1000), p-p65 (Cell Signaling, #3033, 1:1000), p65 (Cell Signaling, #8242, 1:1000), PAR2 (Abcam, ab180953, 1:10,000), PKC-γ (Abcam, ab71558, 1: 2000), PKA (Cell Signaling, #4782, 1:1000) and TRPV1 (Abcam, ab6166, 1:1000) for 12 h at 4°C. Following the incubation with the secondary antibody linked to HRP (Abcam, ab96899, 1:10,000) for 4 h at room temperature, protein was measured by ECL kit (Thermo Scientific, China) and Bio-Rad XR gel imaging analysis system (Bio-Rad, USA).
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