T‐bet, GATA‐3, RORγt, and Foxp3 proteins were extracted using RIPA buffer. Then they were separated on a 10% SDS‐polyacrylamide gel (Sigma‐Aldrich) based on their molecular weights and transferred to a polyvinylidene difluoride (PVDF) membrane (Pharmacia, France) that was blocked with TBS‐Tween‐20 buffer containing 3% BSA for 2 h at room temperature. After washing, overnight incubation at 4°C was done with primary antibodies (1:5000 diluted, Abcam) on the PVDF membrane. Then, after washing, incubation with the secondary horse radish conjugated antibodies (1:5000 diluted, Abcam) was performed for 2 h at room temperature. To detect immunoreactivity, a chemiluminescent substrate was used according to the manufacturer's instructions. A Western blot test was performed in triplicate.
Sds polyacrylamide gel
Manufactured by Merck Group
Sourced in United States
SDS-polyacrylamide gel is a laboratory equipment used to separate and analyze proteins based on their molecular weight. It is a type of gel electrophoresis technique that utilizes sodium dodecyl sulfate (SDS) to denature proteins and polyacrylamide as the supporting matrix.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Abcam (2 mentions)
Tween 20, by Merck Group (2 mentions)
Gelatin, by Merck Group (2 mentions)
Extraction buffer, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
Skim milk, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti gpx1, by Abcam (1 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l hrp, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Irdye 800cw donkey anti mouse, by LI COR (1 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Odyssey clx machine, by LI COR (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
M6818, by Merck Group (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
11 protocols using sds polyacrylamide gel
1
Transcription Factor Protein Analysis
2
Caspase Protein Expression Analysis
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Western blot analysis. The cells were lysed in extraction buffer (Sigma-Aldrich) containing 31.25 mM Tris-HCl (pH 6.8), 1% sodium dodecyl sulfate (SDS), 10% glycerol and 2.5% mercaptoethanol, and the whole-cell lysates were subjected to separation using 10% SDS-polyacrylamide gel (Sigma-Aldrich) electrophoresis. The size-fractionated proteins on the gel were then transferred onto a nitrocellulose membrane (GE Healthcare Life Sciences), and the membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween 20 (Sigma-Aldrich), and was incubated with the following primary antibodies: Goat polyclonal anti-caspase-7 (cat. no. sc-22179), anti-caspase-8 (cat. no. sc-6136) and anti-caspase-9 (cat. no. sc-22182), purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Following washing once with PBS, the membrane was incubated with a horseradish peroxidase-conjugated mouse anti-goat IgG secondary antibody (cat. no. sc-2354; Santa Cruz Biotechnology, Inc.). The protein band of interest was detected using enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Shanghai, China) (20) .
Statistical analysis. Cell viability was expressed as the mean ± standard deviation. Statistical comparisons were made between the control and treatment groups using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Statistical analysis. Cell viability was expressed as the mean ± standard deviation. Statistical comparisons were made between the control and treatment groups using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
3
Western Blot Analysis of Antioxidant Enzymes
4
Affinity Purification of MeCP2 from Rat Brain
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This assay performed as described previously (Connelly et al., 2020 (link))(Piccolo et al., 2019 (link)). PCR-generated, biotin end-labeled 147 bp DNA probes (2 μg) were coupled to M280-streptavidin Dynabeads according to the manufacturer’s instructions (Invitrogen). All cytosines in the probes were either non-methylated or methylated and only occurred in a single sequence context (CG, CAC or CAT), sequences in the Key resources table . Bead-DNA complexes were then co-incubated with 20 μg of rat brain nuclear protein extract (Mellén et al., 2017 (link)) for 1.5 hours at 4◦C. Following extensive washing, bead-bound proteins were eluted using Laemmli buffer (Sigma) and resolved on a 4%–15% SDS-polyacrylamide gel (NEB). The presence of MeCP2 was assayed by western blot using MeCP2 (Sigma M6818; RRID:AB_262075 ) diluted 1:1000; with secondary detection employing Li-COR IRDye 800CW Donkey anti-Mouse (926-32212) diluted 1:10,000, then scanned using a LI-COR Odyssey CLx machine.
5
Immunoblotting Quantification of VEGF Protein
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The immunoblotting was performed as previously described (22 (link)). Briefly, aliquots of the protein samples (30 μg) were separated on an SDS-polyacrylamide gel (Sigma-Aldrich) and were transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA solution [pH 7.4, 0.05% Tween-20 (Amresco LLC) in PBS] for 1 h at room temperature. Anti-VEGF antibody (1:500) was added to the membranes (Millipore, Billerica, MA, USA) for 1 h at room temperature. Membranes were probed with goat anti-rat monoclonal horseradish peroxidase-conjugated secondary antibody (Amersham Life Science, Arlington Heights, IL, USA). The signals were detected by using the enhanced chemiluminescence method (Amersham Life Sciences) and were exposed to X-ray film for autoradiography. The membranes were stripped with a stripping buffer for 15 min at room temperature and immunoblotted with a rabbit anti-mouse actin monoclonal antibody (Santa Cruz Biotechnology, Inc.). The images were scanned and the signal intensity was quantified with Image J software (NIH, Bethesda, MD, USA).
6
Western Blot Analysis of CDK6 Protein
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Cells were lysed using mammalian protein extraction reagent RIPA (Beyotime china) supplemented with protease inhibitors cocktail (Roche, Switzerland) and PMSF (Roche, Switzerland). Protein concentration was measured with the Bio-Rad protein assay kit. 50 μg protein extractions were separated by 12% SDSpolyacrylamide gel electrophoresis (SDS-PAGE), then transferred to 0.22 μm nitrocellulose membranes (Sigma-Aldrich. USA) and incubated with specific antibodies. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad). GAPDH was used as control. GAPDH antibody was purchased from sigma-Aldrich (USA), and CDK6 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
7
Protein Extraction and Quantification
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To prepare total protein from tissues and cells, cartilage obtained at 4 or 8 weeks after treatment in vivo was pulverized in liquid nitrogen and chondrocytes treated with JEZTC or GA followed by induction with IL-1β were washed with cool PBS, then lysis buffer (Sigma-Aldrich, USA) containing a protease inhibitor mixture was used for protein extraction. Equal amounts of protein (60 µg) were separated by SDS-polyacrylamide gel (Sigma, USA), and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). PVDF membranes were blocked with 5% nonfat milk for 1h, and then incubated with primary antibodies (Abcam, UK) of P38(1:1000), p-P38 (1:800), Akt (1:500), p-Akt (1:1000), NF-κB (1:500) overnight at 4°C. The membranes were then incubated with the secondary antibody (Invitrogen, USA) and visualized using the Odyssey Infrared Imaging System (LI-COR, USA) according to the manufacturer's instructions. Data were normalized by housekeeping protein (GAPDH).
8
In Situ Zymography of Myocardial Gelatinase
9
Western Blot Analysis of CRHR Expression
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Purified B cell protein extracts (20 µg) were subjected to electrophoresis on SDS-polyacrylamide gels (Sigma-Aldrich) that were transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fischer Scientific). After blocking, membranes were incubated with an anti-CRHR antibody (diluted 1/1000, BIOSS USA, Interchim). Then, membranes were washed and incubated with an anti-rabbit IgG-peroxidase conjugate (diluted at 1/3000, Jackson ImmunoResearch, Interchim). To quantify the amounts of CRHR relative to the one of GAPDH (glyceraldehyde 3 phosphate dehydrogenase), blots were stripped with stripping buffer (Thermo Fisher Scientific) and reprobed using an anti-GAPDH antibody (diluted /10000, Sigma-Aldrich) followed by an anti-rabbit IgG-peroxidase conjugate. Immunocomplexes were visualized by chemiluminescence (FX7, Vilbert-Lourmat, Marne la Vallée, France) and signals were analyzed by densitometry (ImageJ®, NIH, USA).
10
Assessing Cellular Oxidative Stress and Apoptosis
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Materials purchased from Sigma Chemical Co. (St. Louis, MO, USA) were thymol, DMSO (dimethyl sulfoxide), DCFH-DA (2′,7′- dichlorofluorescein-diacetate), AO/EB (acridine orange/ethidium bromide) stain, SDS polyacrylamide gels, coomassie brilliant-blue-dye, powdered skim milk, trypan blue, comet assays, low-melting agarose, normal melting agarose, and lysis solution. Ham’s F-12 culture medium, fetal bovine serum (FBS), and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). CellTiter-Glo Luminescent Cell Viability and GSH/GSSG-Glo assays were provided by Promega (Madison, MI, USA). Bax (N-20), Bcl-2 (C-21), Caspase-3 (H-277), Caspase-9 (H-170), and β-actin (AC-15) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Amersham ECL Plus Western Blotting Detection reagents were obtained from GE Healthcare (Piscataway, NJ, USA). Radioimmunoprecipitation assay (RIPA) buffer and a proteinase inhibitor cocktail were from Roche (Mannheim, Germany). Molecular weight marker BenchMark Pre-Stained Protein Ladder was from Invitrogen (Grand Island, New York, USA).
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