Cells were harvested on ice by using NETN (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, and 0.5% NP40) buffer supplemented with protease inhibitors, and lysates were incubated with antibodies for overnight at 4°C. Immunocomplexes were precipitated with protein A/ G (Roche Applied Science) for 3 hr at 4°C. The beads were washed four times with NETN buffer. Proteins were then eluted from the beads by boiling in sample buffer for 5 min and then analyzed by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Whole-cell lysates were analyzed with the following antibodies: pcMET (#3077 Cell Signaling), cMET (#8198 Cell Signaling), CD44v6 (BBA13 R&D), pERK 1/2 (GTX59568 #9101 Cell Signaling), ERK 1/2 #4695 Cell Signaling, H3K27me3 ab6002 Abcam, Histone 3 (GTX122148, #9715 Cell Signaling), ß-Actin (sc-47778 Santa Cruz Biotechnology), EZH2 (#3147, #5246 Cell Signaling), pP38 (#9211 Cell Signaling), P38 (sc-7149 Santa Cruz Biotechnology), SULF1 (ab32763 Abcam), and 10E4 (#370255 S, amsbio). The image detection was analyzed by ChemiDoc Touch Imaging System (Bio-Rad) and Image Lab software (Bio-Rad).
Protein a g
Manufactured by Roche
Sourced in Switzerland
Protein A/G is a recombinant protein that contains the binding sites for both Protein A and Protein G. It is commonly used in affinity chromatography for the purification of antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Cytiva (3 mentions)
Ecl system, by Cytiva (2 mentions)
Histone 3, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Sulf1, by Abcam (1 mentions)
P c met, by Cell Signaling Technology (1 mentions)
C met, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using protein a g
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot and Coimmunoprecipitation Protocol
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Cell lysates were made in lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.4, 1% Triton X-100, 10% glycerol and a mixture of protease inhibitors). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and incubated with primary antibodies followed by horseradish peroxidase conjugated secondary antibodies (Amersham Biosciences). Blots were developed using the ECL system (Amersham Biosciences). For coimmunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Roche), resolved by SDS-PAGE and analyzed by immunoblot assay.
3
Protein Extraction and Immunoblotting Protocol
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Cell lysates were made in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.2, 1% NP40, 2 mM EDTA and a mixture of protease inhibitors). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA). Blots were developed using the ECL system (Amersham Biosciences). For coimmunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Roche, Basel, Switzerland), resolved by SDS-PAGE and analyzed by immunoblot assay. All immunoblots were done at least three times using different biological material as sources. Phosphatase Inhibitor Cocktail was purchased from Sigma and used according to the instructions provided.
4
Immunoprecipitation and Immunoblotting Analysis
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Cell lysates were made in lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol and a mixture of protease inhibitors). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and incubated with primary antibodies followed by horseradish peroxidase conjugated secondary antibodies (Amersham Biosciences). Blots were developed using an ECL system (Pierce). For coimmunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Roche), resolved by SDS-PAGE and analyzed by immunoblot assay.
For the denaturing immunoprecipitation, 293T cells were transiently transfected at 60% confluency with retroviral vectors encoding the indicated shRNAs. Cell lysates prepared in lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris–HCl pH 7.2, a protease inhibitors cocktail, 50 mM NaF, 1 mM NaOV, 25 mM beta-phosphoglycerate lysis) underwent anti-NEMO immmunoprecipitation for 4 hrs at 4 °C. Immunocomplexes were then dissociated in PBS containing 1% SDS at 37 °C for 1 h, diluted 10-fold and subjected to a second round of immunoprecipitation by anti-ubiquitin antibody. Immunocomplexes were then washed three times, resolved on 10% SDS-PAGE and immunoblotted as indicated.
For the denaturing immunoprecipitation, 293T cells were transiently transfected at 60% confluency with retroviral vectors encoding the indicated shRNAs. Cell lysates prepared in lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris–HCl pH 7.2, a protease inhibitors cocktail, 50 mM NaF, 1 mM NaOV, 25 mM beta-phosphoglycerate lysis) underwent anti-NEMO immmunoprecipitation for 4 hrs at 4 °C. Immunocomplexes were then dissociated in PBS containing 1% SDS at 37 °C for 1 h, diluted 10-fold and subjected to a second round of immunoprecipitation by anti-ubiquitin antibody. Immunocomplexes were then washed three times, resolved on 10% SDS-PAGE and immunoblotted as indicated.
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