Total protein extraction of NK cells, NK-92 cells lines and tumor tissues was performed using RIPA lysis buffer (Beijing Solarbio Science and Technology, Beijing, China). Protein samples were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) and blocked with tris buffered saline tween (TBST) buffer containing 5% non-fat milk at room temperature for 1 h. The membranes were incubated with primary antibodies rabbit anti-RUNX3 (1:1000; Cell Signaling Technology, Boston, MA, USA) and anti-NKp46 (1:1000; Bioworld Technology Inc. Minneapolis, MN, USA) at 4 °C overnight, followed by rinsed with TBST thrice, and then incubated with goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h under a shaker. Protein bands were visualized using an enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA, USA). Band intensities were standardized to β-actin (Sigma-Aldrich) as a loading control.
Enhanced chemiluminescence reagent
Manufactured by Beckman Coulter
Sourced in United States
The Enhanced chemiluminescence reagent is a laboratory product designed to facilitate chemiluminescence detection and analysis. It provides the necessary components to generate and amplify luminescent signals, enabling sensitive and quantitative measurements in various biochemical and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab85163, by Abcam (3 mentions)
Polyvinylidene uoride membrane, by Merck Group (3 mentions)
Ab8245, by Abcam (3 mentions)
Primary anti active β catenin antibody, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Rorγt, by Cell Signaling Technology (1 mentions)
Foxp3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
8 protocols using enhanced chemiluminescence reagent
1
Protein Extraction and Western Blot Analysis of NK Cells
2
Protein Expression Analysis in Fibroblasts
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Total protein was extracted from cultured fibroblasts using RIPA lysis buffer (Pierce; Thermo Fisher Scientific, Inc.). The protein quantification was performed using bicinchoninic acid method (Thermo Fisher Scientific, Inc.). Equal amounts of protein (40 µg) were electrophoresed via 10% SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes (MilliporeSigma). After blocking in 5% skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibody (1:1,000) overnight at 4˚C and horseradish peroxidase-conjugated IgG secondary antibody (1:5,000) for 1 h at room temperature. The primary antibodies against Col I (cat. no. ab6308), OPN (cat. no. ab8448), Runx2 (cat. no. ab76956), VDR (cat. no. ab134826) and GAPDH (cat. no. ab8245) and secondary antibody (cat. no. ab8245) were all purchased from Abcam. Densitometric analysis was processed using enhanced chemiluminescence reagent (Beckman Coulter, Inc.). Protein bands were semi-quantified using Image J software (version 1.8.0; National Institutes of Health).
3
Western Blot Analysis of Active β-Catenin
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We used lysis buffer to extract protein, which was isolated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was then transferred to a polyvinylidene fluoride membrane, blocked with a primary anti‐active β‐catenin antibody (Millipore, Bedford, MA) overnight at 4°C and incubated with an anti‐mouse horseradish peroxidase‐conjugated secondary antibody (Cell Signaling Technology, Boston, MA) after being washed with Tris‐buffered saline at 37°C for 1 hour. Protein quantification was performed using an enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA). GAPDH was used as a loading control.
4
Western Blot Analysis of T Cell Proteins
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Total protein was extracted from cultured CD4+T cells and PBMCs using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene fluoride (PVDF) membranes. Following blocking with 5% skimmed milk for 2 h at room temperature, these membranes were incubated with primary antibodies against SOCS3 (#52,113), Foxp3 (#12,653), STAT3 (#12,640), RORγt (#16,540) (all from Cell Signaling Technology, Boston, MA, USA;1:1,000 dilution) overnight at 4˚C. The next day, membranes were incubated at room temperature for 2 h with FITC-labeled IgG secondary antibodies (#7074; 1:2,000 dilution; Cell Signaling Technology). The enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA, USA) was used to detect the protein bands, and the protein expression levels were quantified using ImageJ software (version 1.6.0; National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Active β-catenin
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Total protein was extracted by lysis buffer and isolated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked with primary anti active β-catenin antibody (Millipore, Bedford, MA, USA) overnight at 4 °C and incubated with anti-mouse horseradish-peroxidase (HRP) conjugated secondary antibody (Cell Signaling Technology, Boston, MA, USA) after washed with Tris-buffered saline containing 10 mM Tris–HCl, 50 mM NaCl and 0.25% Tween 20 at 37 °C for 1 h. Protein quantification was performed using an enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA, USA). GAPDH was used as a loading control.
6
Protein Quantification in Tissues and Cells
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Total protein in tissues, cells and exos was obtained by radio-immunoprecipitation assay lysate containing protease inhibitors, separated by 10% sodium lauryl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene uoride membrane (Millipore, MA, USA) and sealed with 5% skim milk. The corresponding primary antibodies EDNRA (1:1000, ab85163) and GAPDH (1:1000, ab8245, Abcam), as well as the corresponding secondary antibody were incubated with the membrane.
The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
7
Quantitative Protein Analysis via RIPA
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Total protein in tissues, cells and exos was obtained by radio-immunoprecipitation assay lysate containing protease inhibitors, separated by 10% sodium lauryl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene uoride membrane (Millipore, MA, USA) and sealed with 5% skim milk. The corresponding primary antibodies EDNRA (1:1000, ab85163) and GAPDH (1:1000, ab8245, Abcam), as well as the corresponding secondary antibody were incubated with the membrane. The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
8
Protein Quantification in Tissues and Cells
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Total protein in tissues, cells and exos was obtained by radio-immunoprecipitation assay lysate containing protease inhibitors, separated by 10% sodium lauryl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene uoride membrane (Millipore, MA, USA) and sealed with 5% skim milk. The corresponding primary antibodies EDNRA (1:1000, ab85163) and GAPDH (1:1000, ab8245, Abcam), as well as the corresponding secondary antibody were incubated with the membrane.
The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
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