Cells were lysed in RIPA buffer (Sigma) containing Complete-ULTRA protease-inhibitor and PhoStop phosphatase-inhibitor cocktails (Roche), and sonicated with Bioruptor200 (Diagenode) at high frequency, alternating 30 s on/off for 3 min. SDS-PAGE electrophoresis was performed using Bolt 10% Bis-Tris Plus gels (ThermoFisher) in a Novex MiniCell (ThermoFisher). Protein transfer was performed using the semi-dry iBlot2 system (ThermoFisher) and iBlot Transfer Stacks (ThermoFisher). The following primary antibodies were used: Esrrb (1:1000, mouse mAb, Perseus Proteomics); Klf2 (kind gifts from Huck-Hui Ng; 1:500, rabbit serum, Yeo et al., 2014 (link); and Hitoshi Niwa (1:1000, mouse mAb; Yamane et al., 2018 ); Oct4 (1:1000, rabbit mAb, Cell Signaling); P-Y705-Stat3 (1:1000, rabbit mAb, Cell Signaling); αTubulin (1:10000, mouse mAb, Abcam). Detection was achieved using HRP-linked secondary antibodies at 1:10000 against the appropriate species (GE Healthcare) and ECL Plus Western Blotting Detection System (GE Healthcare).
Bioruptor 200
Manufactured by Diagenode
The Bioruptor 200 is a laboratory equipment used for the shearing and fragmentation of DNA, RNA, and proteins. It utilizes high-intensity ultrasonic waves to disrupt the molecular bonds, enabling the sample to be processed into smaller fragments. The Bioruptor 200 is designed to provide consistent and reproducible results for various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Iblot transfer stack, by Thermo Fisher Scientific (3 mentions)
Bolt 10 bis tris plus gel, by Thermo Fisher Scientific (3 mentions)
Iblot2 system, by Thermo Fisher Scientific (3 mentions)
Novex mini cell, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Ecl plus western blotting detection system, by GE Healthcare (3 mentions)
Py705 stat3, by Cell Signaling Technology (2 mentions)
α tubulin, by Abcam (2 mentions)
Complete ultra protease inhibitor, by Roche (2 mentions)
Phostop phosphatase inhibitor cocktail, by Roche (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
6 protocols using bioruptor 200
1
Western Blot Analysis of Pluripotency Factors
2
Protein Extraction and Western Blotting Procedure
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Cells were lysed in RIPA buffer (Sigma) containing Complete-ULTRA protease-inhibitor and PhoStop phosphatase-inhibitor cocktails (Roche), and sonicated with Bioruptor200 (Diagenode) at high frequency, alternating 30 s on/off for 3 min. SDS-PAGE electrophoresis was performed using Bolt 10% Bis-Tris Plus gels (ThermoFisher) in a Novex MiniCell (ThermoFisher). Protein transfer was performed using the semi-dry iBlot2 system (ThermoFisher) and iBlot Transfer Stacks (ThermoFisher). The following primary antibodies were used: Esrrb (1:1000, mouse mAb, Perseus Proteomics); Klf2 (kind gifts from Huck-Hui Ng; 1:500, rabbit serum, Yeo et al., 2014 (link); and Hitoshi Niwa (1:1000, mouse mAb; Yamane et al., 2018 (link)); Oct4 (1:1000, rabbit mAb, Cell Signaling); P-Y705-Stat3 (1:1000, rabbit mAb, Cell Signaling); αTubulin (1:10000, mouse mAb, Abcam). Detection was achieved using HRP-linked secondary antibodies at 1:10000 against the appropriate species (GE Healthcare) and ECL Plus Western Blotting Detection System (GE Healthcare).
3
Chromatin Immunoprecipitation and Re-ChIP Assay
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MIN6 cells were fixed and chromatin DNA-protein was cross-linked with 1% formaldehyde for 10 min, then quenched with 125 mM glycine for 5 min, and finally washed with phosphate-buffered saline. Fixed cells were sonicated with a Bioruptor 200 (Diagenode) and lysed in a RIPA assay buffer containing protease inhibitors. DNA-protein complexes were immunoprecipitated with indicated antibodies or IgG isotype controls and washed. Cross-links were reversed at 65°C for 4 h in Tris buffer containing 5 M NaCl and 0.5 M EDTA. DNA was extracted by phenol/CHCl3 and precipitated with ethanol followed by proteinase K and RNase A treatment. Precipitated DNA and 1% control inputs were analyzed by QPCR.
For Re-ChIP samples, eluates were diluted 10-fold to reduce SDS concentration, and multiple samples were pooled and scaled up 18-fold to provide sufficient material for a second round of immunoprecipitations with a second pull-down antibody. Cross-links were reversed with 0.2 M NaCl at 65°C for 5 h, and the DNA was purified by phenol-chloroform extraction. Promoter DNA precipitate was measured by QPCR and normalized to IgG and input DNA control samples.
For Re-ChIP samples, eluates were diluted 10-fold to reduce SDS concentration, and multiple samples were pooled and scaled up 18-fold to provide sufficient material for a second round of immunoprecipitations with a second pull-down antibody. Cross-links were reversed with 0.2 M NaCl at 65°C for 5 h, and the DNA was purified by phenol-chloroform extraction. Promoter DNA precipitate was measured by QPCR and normalized to IgG and input DNA control samples.
4
Chromatin Immunoprecipitation Assay Protocol
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Chromatin immunoprecipitation (ChIP) assays were performed as previously described (6 (link)). Briefly, MIN6 cells were fixed, and chromatin DNA-protein was cross-linked with 1% formaldehyde for 10 min, quenched with 125 mmol/L glycine for 5 min, and washed with PBS. Fixed cells were sonicated with a Bioruptor 200 (Diagenode) and lysed in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail. DNA-protein complexes were immunoprecipitated with the indicated antibodies or IgG isotype controls and extensively washed. Cross-links were reversed at 65°C for 4 h in Tris buffer containing 5 mol/L NaCl and 0.5 mol/L EDTA. DNA was extracted by phenol/CHCl3 and precipitated with ethanol, followed by proteinase K and ribonuclease A treatment. Precipitated DNA and 1% control inputs were analyzed by qPCR.
5
Cell Lysis and Western Blot Analysis
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Cells were lysed in RIPA buffer (Sigma) containing Complete-ULTRA protease-inhibitor and PhosSTOP phosphatase-inhibitor cocktails (Roche), and sonicated with a Bioruptor 200 (Diagenode) at high frequency, alternating 30 s on/off for 3 min. SDS-PAGE was performed using Bolt 10% Bis-Tris Plus gels (Thermo Fisher) in a Novex Mini-Cell (Thermo Fisher). Protein transfer was performed using the semi-dry iBlot2 system (Thermo Fisher) and iBlot Transfer Stacks (Thermo Fisher). Detection was achieved using horseradish peroxidase-linked secondary antibodies diluted 1:10,000 against the appropriate species (GE Healthcare) and the ECL Plus Western Blotting Detection System (GE Healthcare). See the supplemental experimental procedures for primary antibodies.
6
ChIP Assay for NFATc1, NFATc2, and TFII-I
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