Nuclear/cytoplasmic fractionation assays were conducted with NE‐PER Nuclear and Cytoplasmic Extraction Reagents (78835, Millipore, Burlington, MA, USA). Swan 71 or HTR‐8/SVneo cells were lyzed with a homogenizer in cold cytoplasmic lysis buffer on ice for 10 min. The supernatant was collected by centrifugation at 15 000 × g for 15 min at 4 °C as cytoplasmic proteins. The precipitate was re‐suspended in nuclei lysis buffer and vortexed for 30 min at 4 °C. Then, the supernatant was collected by centrifugation at 15 000 × g for 15 min at 4 °C as nuclear proteins. The cytoplasmic and nuclear proteins were analyzed by Western blotting, with H3 as nuclear marker and β‐Tubulin as cytoplasmic marker.
Ne per nuclear and cytoplasmic extraction reagents
Manufactured by Merck Group
Sourced in United States
The NE-PER Nuclear and Cytoplasmic Extraction Reagents are a set of reagents designed for the extraction and separation of nuclear and cytoplasmic proteins from mammalian cells. The reagents enable the efficient isolation of these cellular fractions for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Polyvinylidene uoride membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Cell Signaling Technology (1 mentions)
Anti lc3 antibody, by Cell Signaling Technology (1 mentions)
Anti p65 antibody, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection system, by Merck Group (1 mentions)
Nlrp3, by Santa Cruz Biotechnology (1 mentions)
Anti atg5 antibody, by Cell Signaling Technology (1 mentions)
Anti caspase 1 antibody, by Cell Signaling Technology (1 mentions)
Anti trif antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g, by Cell Signaling Technology (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
6 protocols using ne per nuclear and cytoplasmic extraction reagents
1
Subcellular Fractionation for Cellular Proteomics
2
Western Blot Protein Analysis
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Cells were harvested at 90% con uence. For regular immunoblots, cell lysates were obtained using RIPA Lysis Buffer (Millipore, MA, USA). Separate nuclear and cytoplasmic protein extraction was performed NE-PER Nuclear and Cytoplasmic Extraction Reagents (Millipore, MA, USA). Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene uoride membranes (Millipore, MA, USA). The membranes were probed with primary antibodies overnight at 4 °C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immune complexes were detected using enhanced chemiluminescence (Cell Signaling Technology, MA, USA). GAPDH was used to correct for differences in loading of the proteins from the control and experimental groups. Detailed antibody information is displayed in Supplementary Data 3.
3
Western Blot Analysis of Inflammasome Proteins
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The cells were lysed in buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail and 1 mM PMSF. The lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer and were subjected to SDS–PAGE. Cytoplasmic extracts and nuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Sigma, 78833). Rabbit monoclonal antibodies against GAPDH, NLRP3, and ASC were purchased from Santa Cruz Biotechnology (CA, USA) and were diluted 1:1000. The anti-ATG5 antibody, anti-LC-3 antibody, anti-Caspase-1 antibody, anti-PCNA, and anti-p65 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and were diluted 1:1000. The anti-TRIF antibody was purchased from Abcam and was diluted 1:1000. Horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, MA, USA) was used as the secondary antibody. Immunoreactive bands were identified using the ECL Western Blotting Detection System (Millipore Corporation, Billerica, MA, USA).
4
Subcellular Fractionation of HCT116 Cells
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HCT116 cells were transfected with the indicated constructs. After 48 h, the cells were treated with MG132 (20 μM) for 6 h. Subsequently, the cells were collected by centrifugation for 5 min at 900 r.p.m. and washed three times with cold PBS. Cytoplasmic and nuclear fractions were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Sigma), following the manufacturer’s procedures.
5
Protein Extraction and Fractionation Protocol
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Total protein was extracted using a lysis buffer, which consisted of 2% Triton X-100 and 0.1% phosphatase inhibitor cocktail (Sigma) in PBS, and lysates were dissolved in Laemmli solution (30 (link)). For subcellular protein fractionations, the NE-PER nuclear and cytoplasmic extraction reagents (Sigma) were used. Immunoblotting was conducted using specific antibodies described in Supplementary Table 3 . Band intensities were determined by a Fusion Solo (Paris, France).
6
Total Protein Extraction and Subcellular Fractionation
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Total protein was extracted using a lysis buffer, which consisted of 2% Triton X-100 and 0.1% phosphatase inhibitor cocktail (Sigma) in PBS, and lysates were dissolved in Laemmli solution (30) . For subcellular protein fractionations, the NE-PER nuclear and cytoplasmic extraction reagents (Sigma) were used. Immunoblotting was conducted using specific antibodies described in Supplementary Table 3. Band intensities were determined by a Fusion Solo (Paris, France).
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