Coimmunoprecipitation and immunoblot analyses were performed as previously described.46 (link) In brief, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) containing protease and phosphatase inhibitor cocktail (Selleck, Houston, Texas, USA). For coimmunoprecipitation, lysates were incubated overnight with ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich), EZview™ Red Anti-HA Affinity Gel (Millipore, Billerica, MA, USA), or Pierce Glutathione Agarose (Thermo Scientific, Rockford, IL, USA), then proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking in TBST containing 5% BSA, the blots were probed with primary antibodies. Determination of the band intensities were performed with ChemiDoc XRS+ Molecular Imager software (Bio-Rad, Philadelphia, PA, USA).
Chemidoc xrs molecular imager software
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc XRS+ Molecular Imager software is a comprehensive imaging and analysis software designed for use with the ChemiDoc XRS+ imaging system. The software provides tools for capturing, processing, and analyzing images of gels, blots, and other samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 affinity gel, by Merck Group (10 mentions)
Coomassie plus protein assay reagent, by Thermo Fisher Scientific (8 mentions)
Ezview red anti ha affinity gel, by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Selleck Chemicals (1 mentions)
Pierce glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Viafect transfection reagent, by Promega (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Selleck Chemicals (1 mentions)
11 protocols using chemidoc xrs molecular imager software
1
Coimmunoprecipitation and Immunoblot Analyses
2
Immunoprecipitation and Immunoblotting Analysis
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HepG2 or HEK293T cells were transfected with the ViaFect Transfection Reagent (Promega, WI, USA) between 24 and 48 h after the transfection of the expression plasmids; the cells were lysed with 50 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, and 1% Nonidet P-40 (NP-40) containing cocktail inhibitors (Millipore, MA, USA). The cell lysates were immunoprecipitated and then incubated with the ANTI-FLAG® M2 Affinity Gel (Sigma, Burlington, MA, USA) overnight. Immunoblotting was performed as described elsewhere. Briefly, the cells were collected and lysed in ice-cold cell lysis buffer for 30 min, with the tubes tapped every 10 min. The protein concentration was quantified by using the Coomassie Plus™ Protein Assay Reagent (Thermo Scientific, Waltham, MA, USA). The band intensities were quantified with ChemiDoc™ XRS + Molecular Imager software (Bio-Rad, CA, USA). The samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The blots were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% skimmed milk and then probed with the relevant antibodies.
3
Immunoprecipitation and Immunoblotting Analysis
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Between 24 and 48 h after transfection of expression plasmids, cells were lysed with 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 1% NP-40-containing cocktail inhibitors (Selleck). Cell lysates were immunoprecipitated, and then incubated overnight with ANTI-FLAG® M2 Affinity Gel (Sigma). Immunoblotting was carried out as previously described (21 (link)). Briefly, cells were collected and lysed in ice-cold cell lysis buffer (20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% NP40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, and 0.5 mM DTT) for 30 min, with tapping of the tubes every 10 min. The protein concentration was quantified by Coomassie Plus™ protein assay reagent (Thermo Scientific). Band intensities were quantified using the ChemiDoc™ XRS+ Molecular Imager software (Bio-Rad). Samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The blots were blocked in Tris-buffered saline (50 mM Tris, 150 mM NaCl) containing 0.1% Tween-20 and 5% skimmed milk, and then probed with the relevant antibodies.
4
Cell Lysis and Immunoprecipitation for Protein Analysis
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The experimental cells were lysed 24 to 48 h after transfection with the expression plasmids using 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% NP-40 containing cocktail inhibitors (Roche, USA). For immunoprecipitation, the lysates were incubated overnight with the anti-FLAG M2 affinity gel (Sigma, USA). Immunoblotting was carried out as described elsewhere (46 (link)). Briefly, the cells were collected and lysed by adding radioimmunoprecipitation assay (RIPA) lysis buffer together with protease/phosphatase inhibitor cocktail on an ice bath for 30 min and by tapping tubes every 10 min. The protein concentration was quantified by the Coomassie Plus protein assay reagent (Thermo Scientific, Rockford, IL, USA). The quantification of immunoblotting band intensity was performed with the ChemiDoc XRS+ Molecular Imager software (Bio-Rad, Philadelphia, PA, USA). The samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk, the blots were probed with relative antibodies.
5
Cell Lysis and Immunoprecipitation Protocol
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Cells were lysed 24–48 h after transfection of expression plasmids using 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40 containing cocktail inhibitors (Roche, USA). For immunoprecipitation, lysates were incubated overnight with ANTI-FLAG® M2 Affinity Gel (Sigma, USA). Immunoblotting was carried out as described previously (Tan et al., 2018a ). Briefly, cells were collected and lysed by adding RIPA lysis buffer together with Protease/Phosphatase Inhibitor Cocktail on ice for 30 min and by tapping tubes every 10 min. Protein concentration was quantified by Coomassie Plus protein assay Reagent (Thermo Scientific, Rockford, IL, USA). The quantification of immunoblotting band intensity was carried out with ChemiDoc XRS+ Molecular Imager software (Bio-Rad, Philadelphia, PA, USA). Samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking in TBS containing 0.1% Tween-20 and 5% skim-milk, the blots were probed with relative antibodies.
6
Cell Lysis and Immunoprecipitation Protocol
7
Immunoprecipitation and Western Blot Analysis
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At 24–48 h after transfection of the expression plasmids, cells were lysed with 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 1% NP-40 containing cocktail inhibitors (Roche). Cell lysates were immunoprecipitated and incubated with ANTI-FLAG® M2 Affinity Gel (Sigma) overnight. Immunoblotting was carried out as described previously.28 Briefly, cells were collected and lysed in ice-cold cell lysis buffer (20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, and 0.5 mM DTT) for 30 min and by tapping the tubes every 10 min. The protein concentrations of the lysates were quantified by Coomassie Plus protein assay Reagent (Thermo Scientific). Quantification of the band intensities was carried out with ChemiDoc XRS+ Molecular Imager software (Bio-Rad). Samples were separated by SDS-PAGE and transferred onto PVDF membranes. The blots were blocked in TBS containing 0.1% Tween-20 and 5% skimmed milk and were then probed with the relevant antibodies.
8
Immunoprecipitation and Western Blot Analysis
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After 30 h post-transfection, cells were lysed in a immunoprecipitation (IP) lysis buffer containing 50 mM Tris (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 100 µM phenylmethylsulfonyl fluoride (PMSF) and complete TM protease inhibitors (Selleck, Houston, USA) for 30 min at 4 °C. Cell lysates were incubated overnight with ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, St. Louis, USA), EZview™ Red Anti-HA Affinity Gel (Millipore, Billerica, USA), or Anti-MYC Affinity Gel (Millipore, Billerica, USA), then proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking in TBST containing 5% BSA, the blots were probed with primary antibodies, and the relative band intensities were determined with ChemiDoc XRS + Molecular Imager software (Bio-Rad, Philadelphia, USA).
9
Co-immunoprecipitation Assay Protocol
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Co-immunoprecipitation was performed as previously described (Liu et al., 2022 (link)). In brief, cells were lysed in the lysis buffer containing protease and phosphatase inhibitor cocktail (Selleck, Houston, Texas, United States). For co-immunoprecipitation, lysates were incubated overnight with ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich). Then, proteins were separated by SDS-PAGE and electro-transferred onto the PVDF membrane. The membrane-containing proteins were blocked for 1 h at room temperature with 5% BSA in PBST, followed by treatment with the indicated primary antibodies overnight at 4°C. Subsequently, blots were incubated with secondary antibody for 1 h at room temperature and visualized via enhanced chemiluminescence reagents (Millipore, Billerica, MA, United States) with ChemiDoc XRS+ Molecular Imager software (Bio-Rad, Philadelphia, PA, United States).
10
Western Blot Analysis of Protein Levels
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We performed western immunoblotting as in previous studies (Tan et al., 2017 (link)). Briefly, ice-cold cell lysis buffer (20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% NP40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT) was used to lyse cells for 30 min with agitation. After extraction, protein levels were quantified with the Coomassie PlusTM protein assay reagent (Thermo Fisher Scientific). Proteins were then loaded and separated via SDS-PAGE, followed by transfer onto PVDF membranes. Blots were blocked using 5% skim milk in TBST, after which appropriate antibodies were used for protein detection. A ChemiDocTM XRS+ Molecular Imager software (Bio-Rad) was used to quantify protein band densities.
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