The dorsal hippocampus was removed and homogenised with 300 μl of homogenisation buffer containing phosphate inhibitor (1:500), from which 30 μl of homogenate was removed and combined with RIPA buffer containing protease and phosphatase inhibitors. The initial homogenate was processed for plasma membrane isolation as per the manufacturer's instructions (BioVision, Milpitas, CA, USA). Protein was loaded into SDS PAGE 10% gels (BioRad). Gels were transferred to PVDF membrane (Thermo Scientific, Waltham, MA, USA). Primary antibodies were left overnight at 4°C for GR (1:1000, Cell Signaling, Beverly, MA, USA), AMPA receptor (AMPAR, subtype 1) (1:1000, Cell Signaling), actin (1:10,000, Sigma-Aldrich, St. Louis, MO, USA), NMDAR 2B (1:1000, Cell Signaling), SGK1 (1:750, Millipore, Billerica, MA, USA), pCREB (1:500, Abcam, Cambridge, MA, USA) and CREB (1:750, Abcam). Anti-rabbit and anti-mouse secondary antibodies were used at 1:20,000 in TBST and then with tertiary streptavidin–horseradish peroxidase (Cell Signaling) for 1 h. SuperSignal West Pico chemiluminescent (Thermo Scientific) was applied. Bands were analysed using Image J software version 1.45 s (NIH, http://imagej.nih.gov/ij/ ).
Sds page 10 gel
Manufactured by Bio-Rad
Sourced in United States
SDS-PAGE (10% gel) is a laboratory instrument used for separating and analyzing proteins based on their molecular weight. It utilizes sodium dodecyl sulfate (SDS) and polyacrylamide gel electrophoresis to achieve this separation process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent, by Thermo Fisher Scientific (1 mentions)
P creb, by Abcam (1 mentions)
Nmdar2b, by Cell Signaling Technology (1 mentions)
Edta free protease inhibitor tablet, by Roche (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Turbo extra sensitive chemoluminescent substrates, by Euroclone (1 mentions)
Liteablot plus, by Euroclone (1 mentions)
Ecl prime western blotting reagent, by GE Healthcare (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Peroxidase conjugated goat igg fraction to mouse igg orrabbit igg, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Smad4, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Proteoprep sample extraction kit, by Merck Group (1 mentions)
10 protocols using sds page 10 gel
1
Hippocampal Protein Quantification
2
Ribosome Fractionation Assay in HEK293T Cells
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HEK293T cells were plated on 150 mm plates, treated with or without 200 nM ISRIB for 20 min, washed twice with ice-cold PBS, collected and centrifuged for 3 min at 800 rcf at 4°C. The pellets were resuspended in ice-cold lysis buffer: 50 mM Tris pH = 7.5, 400 mM KCl, 4 mM Mg(OAc)2, 0.5% Triton X-100 and protease inhibitors (EDTA-free protease inhibitor tablets, Roche, South San Francisco, CA). The lysates were clarified at 20,000×g for 15 min at 4°C and the supernatant was then subjected to a high-speed spin at 100,000×g for 30 min at 4°C to pellet the ribosomes. The supernatants were then loaded on a 5–20% sucrose gradient and centrifuged in a SW55 rotor for 14 hr at 40,000 rpm 4°C. 13 fractions were collected, protein was chloroform-methanol precipitated, resuspended in SDS-PAGE loading buffer and loaded on SDS-PAGE 10% gels (Bio-Rad, Hercules, CA).
3
Protein Analysis Techniques
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Standard techniques for protein analysis included protein quantification using the Bradford assay, protein separation by SDS/PAGE (10% gels) (Bio-Rad Laboratories) in running buffer (25 mM Tris/HCl, 20 mM glycine and 3.5 mM SDS) and transfer on to Immobilon P membranes (Millipore) with transfer buffer [25 mM Tris/HCl, 20 mM glycine and 10% (v/v) methanol]. Membranes were blocked with 5% (w/v) non-fat dried skimmed milk powder in T-TBS (20 mM Tris/HCl, pH 7.6, 137 mM NaCl and 0.2% Tween 20). Blocking solution for detecting eIF2α phosphorylation was 3% (w/v) BSA in PBS. All primary antibodies were used at a 1:1000 dilution in PBS containing 3% (w/v) BSA. Secondary antibodies were diluted in 5% (w/v) non-fat dried skimmed milk powder in T-TBS: HRP (horseradish peroxidase)-conjugated anti-mouse and anti-rabbit were diluted 1:10000 and HRP-conjugated anti-goat was diluted 1:50000. The HRP signal was developed using the LiteAblot PLUS and TURBO extra sensitive chemoluminescent substrates (Euroclone) and exposed to autoradiographic films (Santa Cruz Biotechnology) or revealed by using the ChemiDoc™ MP System (Bio-Rad Laboratories). Densitometry was performed on at least three independent experiments using ImageJ software (NIH).
4
Ovarian Protein Expression Analysis
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Western blot analysis of PPARγ, COX-2 and StAR was performed. Whole ovarian tissues were
homogenized, sonicated and boiled for 5 min in SDS sample buffer. The samples containing
20 µg of protein were electrophoresed on SDS-PAGE 10% gel (Bio-Rad), and
proteins were transferred onto polyvinylidene fluoride membranes (Bio-Rad). Membranes were
blocked with 5% blocking buffer (Wako Chemicals, Osaka, Japan) for 1 hr at room
temperature and then incubated with primary antibodies: anti-PPARγ (1:500), anti-COX-2
(1:400), anti-StAR (1:2,000) or anti-β-actin (1:5,000) overnight at 4°C. After washing,
the membranes were incubated with peroxidase-conjugated goat IgG fraction to mouse IgG or
rabbit IgG (1:40,000, GE Healthcare, Buckinghamshire, U.K.) for 2 hr at room temperature.
Immunoreactive proteins were detected with ECL Prime Western Blotting Reagent (GE
Healthcare). The signal was analyzed with an ImageQuant LAS 4000 digital imaging system
(GE Healthcare).
homogenized, sonicated and boiled for 5 min in SDS sample buffer. The samples containing
20 µg of protein were electrophoresed on SDS-PAGE 10% gel (Bio-Rad), and
proteins were transferred onto polyvinylidene fluoride membranes (Bio-Rad). Membranes were
blocked with 5% blocking buffer (Wako Chemicals, Osaka, Japan) for 1 hr at room
temperature and then incubated with primary antibodies: anti-PPARγ (1:500), anti-COX-2
(1:400), anti-StAR (1:2,000) or anti-β-actin (1:5,000) overnight at 4°C. After washing,
the membranes were incubated with peroxidase-conjugated goat IgG fraction to mouse IgG or
rabbit IgG (1:40,000, GE Healthcare, Buckinghamshire, U.K.) for 2 hr at room temperature.
Immunoreactive proteins were detected with ECL Prime Western Blotting Reagent (GE
Healthcare). The signal was analyzed with an ImageQuant LAS 4000 digital imaging system
(GE Healthcare).
5
Affinity Purification of PHB1 and PHB2
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This assay was performed by means of FL3-Affigel as described previously [7 (link)]. One hundred million H9c2 cells were washed in PBS and lysed in 2 mL of a lysis buffer consisting of 50 mM Tris-HCl pH 8.0, 120 mM NaCl, 1% NP-40, 5 mM dithiothreitol (DTT), 200 μM Na3VO4, 25 mM NaF, and a protease inhibitor cocktail (Roche Diagnostics, Switzerland). Cellular debris were removed by centrifugation at 10 000 × g for 30 min. Five hundred micrograms of total protein extract was incubated for 12 h at 4°C with 40 μL of FL3-Affigel, negative control (NC)-coupled beads, or uncoupled Affi-Gel beads. The beads were extensively washed with the lysis buffer, and the bound proteins were eluted and reduced in a sample buffer consisting of 63 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, a trace of bromophenol blue (0.05%), and 200 mM DTT for 30 min at 65°C. After cooling on ice, each sample was alkylated with a final concentration of 150 mM iodoacetamide for additional 30 min. The proteins were separated by SDS-PAGE (10% gel; Bio-Rad Laboratories, USA) and western blot analyses were performed using anti-PHB1 and anti-PHB2 antibodies.
6
Smad4 Expression Analysis in SMSCs
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SMSCs were harvested and washed with precooled PBS. Cells were lysed with the RIPA buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1% nonidet-P40, 1% sodium deoxycholate, and 0.1% SDS). The protein concentration was determined with the BCA kit (Thermo Scientific). Thirty micrograms of protein was loaded in each lane of the SDS/PAGE (10% gel) (Bio–Rad) and then transferred on to the PVDF membrane. The membrane was blocked with 5% BSA for 1 h at room temperature and then incubated with the indicated primary antibodies against Smad4 (Abcam) and β-actin (Santa Cruz). The membrane was then incubated with HRP–conjugated secondary antibodies for 1 h at room temperature. The band was visualized by putting ECL at the top and bottom.
7
Western Blot Analysis of Autophagy and Inflammation Markers
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Total protein was extracted from tissues and NSCLC cells using ProteoPrep Sample Extraction kit (Sigma-Aldrich; Merck KGaA). The QuantiPro™ BCA Assay kit (cat. no. QPBCA; Sigma-Aldrich; Merck KGaA) was used to measure protein concentration. Protein samples (30 µg) were separated via SDS-PAGE (10% gel; Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes. The membranes were then blocked with 5% non-fat milk in TBS with 0.05% Tween-20 for 1 h at room temperature. After the membranes were incubated with primary antibodies at 4˚C overnight, secondary antibodies were added and incubated with the membranes at room temperature for 1 h. The bands were visualized with an ECL Western Blotting substrate kit (cat. no. K820; BioVision, Inc.) and the protein expression was quantified using ImageJ v1.8.0 software (National Institutes of Health) with GAPDH as the loading control. The primary antibodies used included: Mouse anti-IL-1β (1:1,000; cat. no. 12242; Cell Signaling Technology, Inc.), rabbit anti-LC3B (1:1,000; cat. no. ab51520; Abcam), rabbit anti-P62 (1:10,000; cat. no. ab109012; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). The corresponding secondary antibodies included: Anti-mouse IgG HRP-conjugated antibody (1;2,000; cat. no. 7076; Cell Signaling Technology, Inc.) and goat anti-rabbit IgG HRP-conjugated antibody (1:20,000; cat. no. ab205718; Abcam).
8
Western Blot Analysis of Protein Samples
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Cell samples were extracted in lysis buffer containing 1% Triton X-100, 75 mM NaCl, 5 mM Tris (pH 7.4), 0.5 mM orthovanadate, 0.5 mM EDTA, 0.5 mM EGTA, 0.25% NP-40 and protease inhibitors. Total protein concentrations in each sample were determined using the BCA (Pierce Biotechnology). Equal amounts of lysate (25–50 μg) were separated on a SDS/PAGE (10% gel; BioRad Laboratories). Proteins were electrophoretically transferred to nitrocellulose membranes, which were then stained with Ponceau Red to verify even transfer. Membranes were blocked in 5% powdered milk resuspended in TBS and then incubated with appropriate antibodies. Secondary antibodies were goat anti-rabbit or anti-mouse coupled to HRP (horseradish peroxidase; Pierce Biotechnology). Blots were developed using SuperSignal reagents (Pierce Biotechnology), exposed to X-ray film and immunoreactive bands were quantified using the ImageJ software.
9
Protein Expression Analysis by Western Blot
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Cells were washed and lysed in lysis buffer (10 mM KCl, 20 mM HEPES, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) containing protease and phosphatase inhibitors. After incubation on ice for 20 min, the lysate was centrifuged at 12000 r.p.m. for 20 min at 4°C, and the supernatant was collected. The protein concentration was assessed using a standard BCA assay (Beyotime). For each sample, 60 μg of protein was separated on SDS-PAGE (10% gel) (Bio–Rad), transferred on to a PVDF membrane (Cat#: 3010040001; Roche Applied Science, Mannheim, Germany). After blocking with 5% milk in TBST (0.1% TBS-Tween 20) at room temperature for 1.5 h, the membrane was washed three times with TBST and incubated with monoclonal mouse anti-TINCR polyclonal antibody (1:2000, Abcam, Cambridge, MA, U.S.A.) or monoclonal rabbit anti-β-actin antibody (1:2000, Abcam, Cambridge, MA, U.S.A.) overnight at 4°C. After washing with PBS for 10 min, the membrane was incubated with mouse anti-rabbit secondary antibody (1:20000, Abcam, Cambridge, MA, U.S.A.) for 1 h at room temperature. After washing with PBS for 15 min, protein bands were detected using the ECL Western Blotting Kit (Pierce Chemical, Rockford, IL, U.S.A.). The relative protein expression was analyzed by Image-Pro Plus software 6.0, represented as the density ratio compared with β-actin.
10
Western Blot Protein Analysis Protocol
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Cells were resuspended in ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) supplemented with a cocktail of protease/phosphatase inhibitors (Thermo Fisher Scientific, Inc.). Cells were further incubated on ice for 10 min and centrifuged at 14,000 × g at 4°C for 20 min. Subsequent to determination of the protein concentration using Bio-Rad Protein Assay (catalog no., 5000006JA; Bio-Rad Laboratories, Inc., Hercules, CA, USA), the proteins in each sample were resolved on SDS-PAGE (10% gel; Bio-Rad Laboratories, Inc.) and transferred onto polyvinylidene difluoride membranes (Merck KGaA). The membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and blocked with Blocking One-P (Nacalai Tesque, Inc.) at room temperature. The membranes were incubated with respective primary antibodies against different targets at 4°C overnight. Subsequent to incubation with primary antibodies, secondary antibodies were added and incubated for 1 h at room temperature. Subsequent to incubation with secondary antibodies, signals were detected with ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little Chalfont, UK).
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