For the in vitro caspase-1 and IL-18 analysis, 40 μl of supernatant was loaded onto 4–12% Bis-Tris SDS gel (NuPAGE Novex, Life Technologies) after denaturing the proteins in Laemmli buffer. Gels were run in MOPS SDS buffer (NuPAGE MOPS SDS running buffer (20×), Life Technologies). Magic Mark XP Western Standard (Invitrogen) was used as a molecular weight marker. Proteins were transferred onto PVDF membranes in Tris-glycine buffer (20% methanol, Tris-glycine (10×), Bio-Rad). Membranes were blocked in 10% non-fat dry milk (TBS (20×) + 0.1% Tween 20) or 10% bovine serum albumin (TBS = 0.1% Tween 20) for the biotin-labeling experiments. Anti-caspase-1 and anti-IL-18 antibodies (1:1,000) were used to probe gels, and respective secondary antibodies (1:10,000) were subsequently added. Membranes were rinsed and washed 3 times with TBS + 0.1% Tween-20 between each of the incubation steps. Enhanced chemiluminescence solution (Amersham, GE Life Sciences) was used to detect labeled proteins and blots were developed using HyBlot CL autoradiography film (Denville Scientific Inc.) on a Konica Minolta SRX-101A film processor (Konica Minolta Medical Imaging U.S.A., Inc.).
Magic mark xp western standard
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Magic Mark XP Western Standard is a pre-stained protein molecular weight marker for use in western blotting applications. It provides an easy-to-use reference for estimating the molecular weights of proteins in SDS-PAGE gels.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (4 mentions)
Ecl prime western blotting detection system, by GE Healthcare (4 mentions)
Phosphatase inhibitor, by Roche (3 mentions)
Enhanced chemiluminescence solution, by GE Healthcare (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage novex, by Thermo Fisher Scientific (1 mentions)
Tris glycine buffer, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ecl plus kit, by Cytiva (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
X omat ls film, by Kodak (1 mentions)
Fibronectin, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Gapdh and β actin, by Abcam (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
8 protocols using magic mark xp western standard
1
Western Blot Analysis of Caspase-1 and IL-18
2
Western Blot Analysis of Spinal Cord Proteins
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The homogenates of spinal cord were used for western blot assay. Forty micrograms of protein per animal was resolved on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The samples were electrophoresed in a 10% polyacrylamide gel. Protein samples were boiled in 2.5% sodium dodecyl sulfate and 5% β-mercaptoethanol. A total of 20 µg protein samples and markers (MagicMark XP Western Standard; Invitrogen, Carlsbad, CA, USA) of each group were electrophoresed at 20 mA for 90 minutes. Electrophoresis buffer was prepared by 25 mM tris(hydroxymethyl)aminomethane, 250 mM glycine and 0.1% sodium dodecyl sulfate. Proteins were transferred onto polyvinylidene fluoride membrane (LC2002; Invitrogen) using transmembrane buffer and 10% methanol. The membrane was incubated with rabbit anti-p-ERK, p-Akt, p-JNK and GAPDH polyclonal antibodies (1:1,000; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) at room temperature for 1 hour, washed with PBS, and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:1,000; Beijing Biosynthesis Biotechnology Co., Ltd.) at room temperature for 90 minutes. The samples were visualized with ECL Plus Kit (Amersham Bio-sciences, Piscataway, NJ, USA). Optical density values were analyzed using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Protein Detection Protocol
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Cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using Magic Mark XP Western Standard (Invitrogen). After blocking with skim milk for 2 h, the membrane sections were incubated with the primary antibody and the corresponding secondary antibody. Primary antibodies were used at a dilution of 1:1000. Secondary antibodies (peroxidase-labeled anti-mouse and anti-rabbit antibodies) were used at a dilution of 1:5000. Finally, bound antibodies were captured by chemiluminescence using the ECL Prime Western blotting (WB) detection system (GE Healthcare), and luminescent images were analyzed with a Lumino Imager (LAS- 4000 mini; Fuji Film Inc.).
4
Quantitative Western Blot Analysis
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For the western blot analysis, 0.3 μg of purified P7 was loaded on to a 12% SDS-PAGE gel. Identical loadings were performed for each of the five Mabs. After gel separation, the proteins were transferred onto a nitrocellulose transfer membrane (BA-S 85 by OPTITRAN) and stained with 0.1% Ponceau S (Sigma-Aldrich, St. Louis, MO) in 5% glacial acetic acid to visualize the transferred bands. The membranes were then cut into five sections for each of the Mabs. The membranes were blocked in 5% skim milk and 0.01% Tween20 in PBS, and then incubated overnight in the presence of 1 μg/ml of each Mab in PBS and 0.01% Tween20. The membranes were then incubated in the presence of a 1:20000 dilution of goat anti-mouse IgG conjugated with HRP (horse radish peroxidase) added to 3% skim milk and 0.01% Tween20 in PBS. To develop the Western Blot Marker (Magic Mark XP Western Standard by Invitrogen) the 1 μl of Precision Protein StrepTactin-HRP conjugated (Bio-Rad Life Sciences, Hercules, CA) was added to each of 10 ml of secondary antibody solution. The membranes were incubated in Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL) and then Kodak X-OMAT LS film was exposed to visualize the HRP luminescence.
5
Protein Extraction and Western Blot Analysis
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Cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). The proteins in lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using Magic Mark XP Western Standard (Invitrogen). After being blocked with skim milk for 2 h, the membrane sections were incubated with the primary antibody and the corresponding secondary antibody. Primary antibodies were used at a dilution according to the instructions, while secondary antibodies were used at a dilution of 1:5000. Finally, bound antibodies were detected by chemiluminescence using the ECL Prime Western blotting detection system (GE Healthcare), and images were analyzed with a Lumino Imager (LAS- 4000 mini; Fuji Film Inc.).
6
Western Blot Analysis of Protein Lysates
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Cells were lysed in a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS with complete protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were separated by 10% SDSPAGE and were transferred to a polyvinylidene difluoride membrane. SDSPAGE gels were calibrated using Magic Mark XP Western Standard (Invitrogen). Primary antibodies were used at a dilution of 1:1,000. Secondary antibodies (peroxidase-labeled anti-mouse and anti-rabbit antibodies) were used at a dilution of 1:5,000. Bound antibodies were visualized by chemiluminescence using the ECL Prime Western blotting (WB) detection system (GE Healthcare), and luminescent images were analyzed with a Lumino Imager (LAS- 4000 mini; Fuji Film Inc.).
7
Western Blot Analysis of Cellular Signaling Pathways
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As described previously,25 (link) whole-cell lysates were prepared after incubation with RIPA buffer (Sigma) on ice for 10 min. Samples were heated at 95 °C for 5 min and subjected for 10 min to centrifugation at 10,000 × g. Cell lysates were separated by 10% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using Magic Mark XP Western Standard (Invitrogen). SDS-PAGE was performed, and proteins were detected using their respective antibodies. Bound antibodies were visualised by chemiluminescence using the ECL Prime Western blotting (WB) detection system (GE Healthcare), and luminescent images were analysed with a Lumino Imager (LAS-4000 mini, Fuji Film Inc.). Each experiment was repeated at least three times. The primary antibodies are as follows: E2F7 (Abcam), cyclin D1, cyclin E, CDK2 and CDK4 (Cell Signaling Technology), N-cadherin, E-cadherin, Fibronectin, Vimentin and MMP9 (Cell Signaling Technology), β-catenin (Abcam), EZH2 and PTEN (Abcam), H3K27me3 (Cell Signaling Technology), AKT, p-AKT at Ser473, mTOR and p-mTOR at Ser2448 (Cell Signaling Technology) and GAPDH and β-actin (Abcam).
8
Western Blot Analysis of Cell Cycle Regulators
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Cells were lysed in a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) with complete protease inhibitor cocktail (Roche, Basel, Switzerland), and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using MagicMark XP Western Standard (Invitrogen). Then, membranes were incubated with a primary antibody against human E2F5 (1:1000; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000; Beyotime, Shanghai, China), CDK2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDK4 (1:500; Santa Cruz Biotechnology), Cyclin D1 (1:500; Santa Cruz Biotechnology), or Cyclin E (1:500; Abcam) at 4°C overnight. After three 10-min washes with tris-buffered saline with Tween 20 (TBST), the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature (1:1000; Santa Cruz Biotechnology). The targeted bands were visualized with enhanced chemiluminescence (ECL).
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