For the Western blot analysis, the total protein was extracted with protein extraction kits (Solarbio, Beijing, China) following the manufacturer’s instructions. Protein was separated by 10% SDS-PAGE. Gels were blotted onto a PVDF membrane (Merck Millipore, Burlington, MA, USA) using a transfer buffer at 64 V for 2 h. The procedure of Western blot analysis was described previously [47 (link)]. The membranes were washed three times with Tris-buffered saline (TBS, 5 min each) and then blocked with 5% nonfat dry milk in TBST (TBS with 0.1% Tween 20) for 1 h at 25 °C with 50 rpm shaking. Then, the membranes were incubated with primary antibody (mouse anti-GFP antibody (1:10,000, (Proteintech, IL, USA)), mouse anti-Bax antibody (1:10,000, Proteintech)) at 4 °C overnight. After washing three for 5 min in TBST, membranes were then incubated with goat anti-mouse IgG (H+L) antibody (Proteintech) as secondary antibody at 1:10,000 dilutions for 2 h, followed by three washes at room temperature. The Protein bands were detected with chemiluminescence horseradish peroxidase substrate (Merck Millipore, Burlington, MA, USA) and photographed following the manufacturer’s instructions.
Chemiluminescence horseradish peroxidase substrate
Manufactured by Merck Group
Sourced in United States
Chemiluminescence horseradish peroxidase substrate is a laboratory reagent used to detect and quantify the presence of horseradish peroxidase (HRP) enzyme in various applications. It emits light upon oxidation catalyzed by HRP, allowing for sensitive detection and measurement of HRP-labeled targets.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Mouse anti gfp antibody, by Proteintech (1 mentions)
Protein extraction kit, by Solarbio (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab10983, by Abcam (1 mentions)
Immobilon p polyvinylidene difluoride membranes, by GE Healthcare (1 mentions)
A5441, by Merck Group (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Complete mini, by Roche (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Pparα, by GeneTex (1 mentions)
Acox1, by Affinity Biosciences (1 mentions)
Srebp1, by Novus Biologicals (1 mentions)
Hmgcr, by Abcam (1 mentions)
5 protocols using chemiluminescence horseradish peroxidase substrate
1
Western Blot Analysis for Protein Detection
2
Protein Extraction and Western Blot Analysis
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Tissue extracts for protein analysis were prepared by homogenization in a buffer containing 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM sodium orthovanadate, 10 mM b-glycerophosphate, 5 mM sodium fluoride and a protease inhibitor cocktail (cOmplete-Mini, Roche, Sant Cugat del Valle `s, Spain). Protein concentration in each sample was determined by the BCA method (Thermo Fisher Scientific). Total protein (30 mg/lane) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide/ bisacrylamide gels using a Mini Trans-Blot kit (Bio-Rad, Hercules, CA, USA) and transferred to Immobilon-P polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK). Membranes were then incubated first with primary antibodies specific for UCP1 (ab10983; Abcam) or b-actin (A5441, Sigma-Aldrich) followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (ab6721, Abcam) or anti-mouse IgG (1721011, Bio-Rad) secondary antibodies. Signals were detected using a chemiluminescence horseradish peroxidase substrate (EMD Millipore) and expressed relative to b-actin signal and total tissue abundance. Digitized images were quantified using the Multi Gauge V3.0 software suite (Fujifilm, Tokyo, Japan).
3
Western Blot Analysis of Lipid Metabolism Proteins
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The samples were lysed using radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using bicinchoninic acid protein assay (G-Biosciences, Maryland Heights, MO, USA). Thirty µg of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked at room temperature for one hour using TBS-T (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, (pH 7.6)) containing 10% skimmed milk, then probed with 1:1000 primary antibodies, such as FAS (Abcam, Cambridge, UK), ACC (Cell signaling, Danvers, MA, USA), SREBP1 (Novus, Centennial, CO, USA), ACOX1, CPT2 (Affinity Biosciences, Pottstown, PA, USA), PPARα (GeneTex, Alton Pkwy Irvine, CA, USA), HMGCR (Abcam, Cambridge, UK) and actin (Abnova, Taipei, Taiwan) at 4 °C overnight. Blots were then washed with TBS-T and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) at room temperature for one hour. Protein bands were visualized using a chemiluminescence horseradish peroxidase substrate (Millipore, Burlington, MA, USA), and the relative signal intensity was quantified using ImageJ software.
4
Protein Expression Analysis of Chidamide and Adriamycin Treated Cells
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Kasumi-1 and HL-60/ADM cells were treated with chidamide, adriamycin, or their combination, and cells were lysed in RIPA buffer (Sigma-Aldrich, USA). Protein levels were determined by western blotting as previously described [1 (link), 44 (link)]. Briefly, whole-cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were probed with an appropriate primary antibody and then incubated with a secondary antibody. The immunoblots were visualized using chemiluminescence horseradish peroxidase substrate (Millipore, USA) and analyzed with the Odyssey Infrared Imaging System (LI-COR Biosciences, USA). Antibodies against acetyl-histone H3 (#8173), DNMT3A (#3598), H3K27me3 (#9733), EZH2 (#5246), Smo (#4940), Gli-1 (#2643), AKT (#4685), p-AKT (#9614) and GAPDH (#5174) were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH was used as a loading control.
5
Protein Expression Analysis in Cancer Cells
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Kasumi-1 cells and HL-60/ADM cells were treated with chidamide, and cells were lysed in RIPA buffer (Sigma-Aldrich, USA). Protein levels were determined by western blot as previously described [1, 52] (link). Brie y, whole cell lysates were separated by SDS-PAGE gel and transferred onto polyvinylidene di uoride (PVDF) membranes (Millipore, USA). Targeted protein was probed with primary antibody, then incubated with the secondary antibody. The immunoblots were visualized using chemiluminescence horseradish peroxidase substrate (Millipore, USA), and analyzed by the Odyssey Infrared Imaging System (LI-COR Biosciences, USA). Antibodies against Acetyl-Histone H3 (#8173), DNMT3A (#3598), H3K27me3 (#9733), EZH2 (#5246), Smo (#4940), Gli-1 (#2643), AKT (#4685), p-AKT (#9614) and GAPDH (#5174) were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH was used as a loading control.
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