Equal amounts of whole cell lysate were mixed with 5 × Laemmeli sample buffer, separated by a SDS 10% polyacrylamide gel (Bio-Rad) and subsequently transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room temperature, membranes were probed for 16 h at 4 °C with the appropriate primary Abs (Santa Cruz Biotechnology, Dallas, CA, USA). After washing, blots were incubated with appropriate HRP-conjugated secondary Ab (Life Technologies, Grand Island, NY, USA) for 2 h at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific). The developed bands were visualized using the ChemiDoc XRS System (Bio-Rad) and the images were captured with the Image Lab Software V3.0. Membranes were stripped and reprobed with β-actin Ab (Sigma-Aldrich) as a loading control.
Sds 10 polyacrylamide gel
Manufactured by Bio-Rad
Sourced in United States
SDS-10% polyacrylamide gel is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It is a type of gel electrophoresis technique that utilizes sodium dodecyl sulfate (SDS) to denature and solubilize proteins, allowing them to migrate through the polyacrylamide gel matrix based on their size.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (12 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (12 mentions)
Chemidoc xrs system, by Bio-Rad (9 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions)
Chemidoc xrs detection system, by Bio-Rad (4 mentions)
β actin antibody, by Merck Group (4 mentions)
Primary abs, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated secondary ab, by Thermo Fisher Scientific (2 mentions)
β actin ab, by Merck Group (2 mentions)
Anti s100a9 antibody, by Santa Cruz Biotechnology (2 mentions)
Il 10, by Santa Cruz Biotechnology (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Kdm6a, by Cell Signaling Technology (1 mentions)
Anti cugbp1, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti ago2 clone 4g8, by Fujifilm (1 mentions)
Anti hur, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ab7091, by Abcam (1 mentions)
13 protocols using sds 10 polyacrylamide gel
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were prepared using 1x RIPA lysis buffer containing 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid, 1 mM EDTA (Millipore, Temecula, CA), and 1x protease inhibitor cocktail. Nuclear and cytoplasmic extracts were prepared using the NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer’s protocol (Pierce, Rockford, IL). Protein extracts were resolved onto SDS-10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). The membranes were blocked with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room temperature and then probed overnight at 4°C with an antibody specific to PU.1 (Cat# MA5-15064, Invitrogen, Carlsbad, CA), Ezh2 (Cat# 166609), IL-10 (Cat# sc-32815), S100A9 (Cat# sc-58706) (Santa Cruz Biotechnology), KDM6A (Cat# 33510S, Cell Signaling Technology, Danvers, MA) antibody. After washing, blots were incubated with the appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific), the bands were visualized using the ChemiDoc XRS System (Bio-Rad), and the images were captured with the Image Lab Software V3.0. The membranes were stripped and reprobed for β-actin (Invitrogen).
3
Immunoblotting Analysis of S100A9 Protein
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Protein extracts or immunoprecipitated protein complexes were resolved by electrophoresis using SDS-10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). Membranes were blocked with 5% milk in Tris-buffered saline/Tween-20 for 1 hr at room temperature, and then probed overnight at 4°C with anti-S100A9 antibody (Cat #sc-58706; Santa Cruz Biotechnology). After washing, blots were incubated with the appropriate HRP-conjugated secondary antibody for 2 hr at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA), the bands were visualized using the ChemiDoc XRS System (Bio-Rad), and the images were captured with the Image Lab Software V3.0. Membranes were stripped and reprobed with b-actin or nucleoporin antibody (Sant Cruz Biotechnology).
4
Immunoblotting Analysis of S100A9 Protein
5
Western Blot Analysis of Protein Expression
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Equal amounts of whole cell lysate were mixed with 5 × Laemmeli sample buffer,
separated by a SDS 10% polyacrylamide gel (Bio-Rad) and subsequently transferred
to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). After
blocking with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room
temperature, membranes were probed for 16 h at 4 ºC with the appropriate primary
Abs (Santa Cruz Biotechnology, Dallas, CA, USA). After washing, blots were
incubated with appropriate HRP-conjugated secondary Ab (Life Technologies, Grand
Island, NY, USA) for 2 h at room temperature. Proteins were detected with the
enhanced chemiluminescence detection system (Thermo Fisher Scientific). The
developed bands were visualized using the ChemiDoc XRS System (Bio-Rad) and the
images were captured with the Image Lab Software V3.0. Membranes were stripped
and re-probed with β-actin Ab (Sigma-Aldrich) as a loading control.
separated by a SDS 10% polyacrylamide gel (Bio-Rad) and subsequently transferred
to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). After
blocking with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room
temperature, membranes were probed for 16 h at 4 ºC with the appropriate primary
Abs (Santa Cruz Biotechnology, Dallas, CA, USA). After washing, blots were
incubated with appropriate HRP-conjugated secondary Ab (Life Technologies, Grand
Island, NY, USA) for 2 h at room temperature. Proteins were detected with the
enhanced chemiluminescence detection system (Thermo Fisher Scientific). The
developed bands were visualized using the ChemiDoc XRS System (Bio-Rad) and the
images were captured with the Image Lab Software V3.0. Membranes were stripped
and re-probed with β-actin Ab (Sigma-Aldrich) as a loading control.
6
Immunoblotting Protocol for Protein Detection
7
Quantitative Protein Expression Analysis
8
Western Blot Analysis of Proteins
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Equal amounts of total protein extract or immunoprecipitated protein complexes (IP) were mixed with 5x Laemmeli sample buffer, separated by a SDS-10% polyacrylamide gel (Bio-Rad) and subsequently transferred to nitrocellulose membranes (Thermo Scientific, Waltham, MA ). After blocking with 5% milk in Tris-buffered saline/Tween-20 for 1 hr at room temperature, membranes were probed overnight at 4°C with the appropriate primary antibody. After washing, blots were incubated with the appropriate HRP-conjugated secondary antibody (Life Technologies, Grand Island, NY) for 2 hr at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Scientific). The developed bands were visualized using the ChemiDoc XRS Detection System (Bio-Rad) and the images were captured with the Image Lab Software V3.0. Membranes were stripped and re-probed with b-actin antibody (Sigma-Aldrich) as a loading control.
9
Western Blot Protein Analysis
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Lysis buffer (Sigma, USA) was added to the cells to isolate total protein. The protein concentration was determined using a bicinchoninic acid assay protein assay kit. Proteins (25 μg/lane) were separated by SDS-10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA). The membrane was blocked with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room temperature, and then probed overnight at 4 °C with the following primary antibodies: anti-IRF6 (1:1000; ab123880; Abcam, Cambridge, MA), anti- KIF20A (1:1000; ab7091; Abcam, Cambridge, MA) (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-GAPDH (ab75478; Abcam, Cambridge, MA). After washing, the blots were incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA). The protein bands were visualized using the ChemiDoc XRS System (Bio-Rad), and the blots were analyzed by Image J software.
10
Western Blot Protein Analysis
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