Whole-cell lysates for western blotting were extracted with RIPA (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease, and phosphatase inhibitor cocktail). Cell lysates (20 ug) were loaded onto 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane and protein expression was depicted with an enhanced chemiluminescence western blot detection kit (Amersham Biosciences). Antibodies used were AZIN1, Antizyme inhibitor 1Polyclonal antibody (Proteintech), GRIA2, AMPA Receptor (GluR2) (E1L8U) Rabbit mAb (Cell Signaling Technology), COG3 polyclonal antibody (Proteintech), V5 Tag Mouse Monoclonal Antibody (Life technologies), and ERK2 (Santa Cruz biotechnology).
Enhanced chemiluminescence western blot detection kit
Manufactured by Cytiva
Sourced in United Kingdom
The Enhanced chemiluminescence Western blot detection kit is a lab equipment product designed for the detection of proteins in Western blot analysis. It uses a chemiluminescent substrate to generate a light signal that can be captured and quantified, allowing for the visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Fractionprep cell fractionation kit, by Abcam (1 mentions)
Nuclear complex co ip kit, by Active Motif (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Ab15048, by Abcam (1 mentions)
Ab41928, by Abcam (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Olig2, by Merck Group (1 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
β3 tubulin, by Merck Group (1 mentions)
L1cam, by Merck Group (1 mentions)
Adar1, by Santa Cruz Biotechnology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
11 protocols using enhanced chemiluminescence western blot detection kit
1
Western Blot Protein Expression Analysis
2
Cell Fractionation and Immunoprecipitation Assay
3
Western Blot Analysis of Protein Expression
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Cells were lysed and sonicated in NETN (0.5% Nonidet P-40, 20 mM Tris [pH 8.0], 50 mM NaCl, 50 mM NaF, 100 µM Na3VO4, 1mM DTT, and 50 µg/mL PMSF) at 4 °C. Crude lysates were cleared by centrifugation at 14,000 rpm at 4 °C for 5 min. After protein quantification, 40 μg of the protein extracts were fractionated using 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% skim milk, the membrane was incubated with primary antibodies overnight at 4 °C. Subsequently, the membrane was incubated with secondary antibodies for 1 h at room temperature. To express the light emission of the proteins, an Enhanced Chemiluminescence Western blot detection kit (Amersham Biosciences, Freiburg, Germany) was used, and the expressed light was captured on Kodak image film. All antibody information is summarized in Supplementary Table S1 .
4
Immunoblot Analysis of Adipogenic Markers
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Equal amounts of protein (40 μg/lane) from the 3T3-L1 cell lysates were resolved by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and immunoblotted with antibodies against JMJD2B, PPARγ, C/EBPα, H3K9me2, H3K9me3, and histone H3. The antibodies for JMJD2B (#ab91549), PPARγ (#ab41928), C/EBPα (#ab15048) were purchased from Abcam (Cambridge, MA, USA). Antibodies for H3K9me2 (#07–441), H3K9me3 (#07–442) and H3 (#06–755) were purchased from Millipore. The proteins were detected using an enhanced chemiluminescence western blot detection kit (Amersham, Uppsala, Sweden).
5
Western Blot Protein Analysis Protocol
6
Western Blotting for Protein Expression Analysis
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Whole-cell lysates for Western blotting were extracted with RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease, and phosphatase inhibitor cocktail). Protein concentrations were determined using bicinchoninic acid (Pierce) assays according to the manufacturer's instructions. Cell lysates (30 µg) were loaded onto 8% or 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane, and protein expression was depicted with an enhanced chemiluminescence Western blot detection kit (Amersham Biosciences). The following antibodies were used: LIFR (1:500, Santa Cruz Biotechnology, sc-659), ZEB1 (1:500, Novus Biologicals, NBP1-05987), and ERK2 (1:2000, Santa Cruz Biotechnology, sc-154), ADAR1 (1:1000, Santa Cruz Biotechnology, sc-271854), ADAR2 (1:1000, Genetex, GTX114237), and GAPDH (1:3000, Santa Cruz Biotechnology, sc-25778).
7
Protein Extraction and Western Blot
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The cells were harvested and washed twice with ice-cold PBS. The resulting cell pellets were lysed with 120 μL of protein lysis buffer (PRO-PREP protein extraction solution (iNtRON Biotechnology Inc.) in ice for 30 min and centrifuged at 13,000×g for 5 min to collect the supernatants. For mouse tissues, even-sized skin samples were first washed with PBS and then lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology Inc.) for 30 min with frequent homogenization. The tissue lysates were centrifuged at 13,000×g for 5 min. Supernatants were collected and protein concentrations were quantified. Aliquots of around 30 μg of protein were boiled for 5 min and resolved in 10% SDS-polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane, blocked with 3% bovine serum albumin solution at 20°C for 1 h, and subjected to incubation with appropriate primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated anti-immunoglobulin G (Pierce, Rockford, IL, USA). The protein bands were detected using an enhanced chemi-luminescence western blot detection kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
8
Western Blot Analysis of Nuclear Proteins
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Western blot analysis was performed as described [28 (link)]. The protein bands were detected using an enhanced chemiluminescence Western blot detection kit (Amersham, Little Chalfont, UK). Nuclear proteins were prepared as previously described [28 (link)]. The membranes were probed with the following primary antibodies: HO-1 (1:1,000), Nrf2 (1:200), TBP (1:2,000), and β-actin (1:5,000). β-actin and TBP were used as each control for equal protein loading in whole cells and nuclear fractions, respectively.
9
Western Blot Protein Analysis Protocol
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NaccSh tissues were homogenized in lysis buffer (20 mM Tris, 5 mM EDTA, and 1% Nonidet P-40 (vol/vol)) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Rockford, IL, USA) and centrifuged at 16,000 × g for 20 min at 4°C. The total protein in the supernatants was quantified by bicinchoninic acid assay, separated by electrophoresis, and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary and secondary antibodies, and the corresponding bands of the proteins of interest were visualized using an enhanced chemiluminescence western blot detection kit (Amersham Biosciences, Piscataway, NJ, USA).
10
Western Blot Analysis of Cell Lysates
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Whole‐cell lysates used for western blot analysis were extracted using RIPA buffer (Biyuntian, Nanjing, China). The cell lysates were loaded onto SDS‐PAGE gels and transferred to polyvinylidene fluoride membranes. Protein expression was determined using an enhanced chemiluminescence western blot detection kit (Amersham Biosciences). Ponceau staining of the uncropped membrane was used as a loading control. Normalisation was performed after the same samples were blotted with antibody against α‐tubulin, AKT or GSK accordingly.
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