Antibodies specific for MEKK2, MEKK3, c-RAF, JNK, ubiquitin and ERK2 were purchased from Santa Cruz Biotechnology. Anti-vinculin antibodies, anti-tubulin antibodies, anti-FLAG antibodies and M2 anti-FLAG affinity gel were purchased from Sigma. Protein A and Protein G agarose beads were purchased from Roche Applied Science. Anti-paxillin antibodies were purchased from BD Biosciences, and anti-Myc tag antibodies were purchased from Millipore. Anti-phospho-c-Jun antibodies, anti-phospho ERK5 antibodies and anti-beta 1 integrin antibodies were purchased from Cell Signaling Technology. Alexa Fluor®-conjugated secondary antibodies were purchased from Invitrogen. Anti-polyubiquitin (K63 linkage-specific) monoclonal antibodies were purchased from Enzo, and linkage-specific K48 anti-ubiquitin antibodies were purchased from Abcam. Fibronectin and JNK inhibitor SP600125 were purchased from Sigma, while MEK5 inhibitor BIX02189 was purchased from Selleck Chemicals.
Protein a and protein g agarose beads
Manufactured by Roche
Protein A and Protein G agarose beads are laboratory reagents used for the purification of antibodies. Protein A and Protein G are bacterial proteins that bind to the Fc region of immunoglobulins. These beads are commonly used in affinity chromatography techniques to capture and purify antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Anti tubulin antibody, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Anti paxillin antibody, by BD (1 mentions)
Anti myc tag antibody, by Merck Group (1 mentions)
Anti vinculin antibody, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
2 protocols using protein a and protein g agarose beads
1
Antibodies and Reagents for Signaling Pathway Analysis
2
Co-immunoprecipitation of Ccp1 and Set2
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Asynchronous cells expressing Ccp1 tagged with GFP-HA fusion (Ccp1-HA) and Set2-FLAG were collected at OD600nm <1.0. Cell extracts were prepared as described previously (57 ), with modifications. Briefly, cells were lysed with Breaking buffer [50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 1 mM MgOAc, 0.1% NP40, 0.5 mM EDTA, 10% glycerol, 2 mM PMSF, supplemented with cOmplete protease inhibitor combination (Roche Diagnostics)] and then homogenized with FastPrep-24 (MP Biomedicals, Santa Ana, CA, USA). Immunoprecipitation was performed at 4°C for 2 h with rotation using 2.4 μg of αFLAG and αHA antibodies prebound for 1 h with a 20-μl mixture of Protein A and Protein G agarose beads (Roche Diagnostics). Samples were washed 5 times with lysis buffer and the bound proteins were eluted by boiling in 2× sample buffer (0.125 M Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) before immunoblotting. For co-immunoprecipitation (CoIP) between Swc2 and Ccp1, Swc2 was tagged with 3× HA (Swc2-HA) and Ccp1 was tagged with GFP (Ccp1-GFP) in the background of Δset2cnp1-1 cells.
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