HEK293T cells were transfected with the indicated expression plasmids for 24 h using JetPRIME transfection reagent (Polyplus, NY). Primary cultures of calvarial osteoblasts were differentiated for 6 days. Cells were lysed in IP buffer (20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 2.5 mM MgCl2, 0.05% NP-40, 1 mM PMSF, and protease inhibitor cocktail (Nacalai Tesque)) and then were incubated on ice for 30 min. Next, the lysed cells were passed through a 29 G needle (Terumo) 6 times. After centrifugation at 14,000×g, the cleared lysates were subjected to immunoprecipitation overnight at 4 °C with anti-HA antibody beads (clone 4B2, Wako Pure Chemical Industries, Ltd.) or anti-OSX-conjugated magnetic beads, which were prepared using 10 μg of OSX antibody (ab22552, Abcam) and 1 mg of beads according to the protocol of the Dynabeads Antibody Coupling Kit (Invitrogen). Then the beads were washed five times with IP buffer, after which proteins were eluted using 2× SDS sample buffer (100 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, and 0.2% bromophenol blue) and were examined by western blotting with the indicated antibodies.
Anti ha antibody beads clone 4b2
Manufactured by Fujifilm
The Anti-HA antibody beads (clone 4B2) are a laboratory tool used for immunoprecipitation and affinity purification. The beads are covalently coupled with a high-affinity monoclonal antibody that specifically binds to the hemagglutinin (HA) epitope tag. This allows for the isolation and capture of HA-tagged proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
29 g needle, by Terumo (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Ab22552, by Abcam (1 mentions)
Dynabeads antibody coupling kit, by Thermo Fisher Scientific (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
Scriptmax thermo t7 transcription kit, by Toyobo (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase a, by Nippon Gene (1 mentions)
Pierce crosslink immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti ha antibody beads clone 4b2
1
Immunoprecipitation of Osteoblast Proteins
2
Quantifying IMP2-Bound Ucp1 Transcripts
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pTD1-Ucp1L, pTD1-Ucp1S, and pTD1-Ucp1C were linearized by NotI digestion and subjected to RNA synthesis using ScriptMAX Thermo T7 Transcription Kit (TOYOBO). The yielded RNAs were collected using a RNeasy Mini Kit (QIAGEN) and used as RNA input for an immunoprecipitation assay, as described below.
HEK293T cells transfected with pcDNA3.1-3×HA, pcDNA3.1-3×HA-IMP2, pcDNA3.1-3×HA-IMP2K438R, or pcDNA3.1-3×HA-IMP2K438Q by the JetPRIME reagent for 24 h were lysed in IP buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM MgCl2, 0.05% NP-40, 1 mM PMSF, and PIC) containing 10 mM nicotinamide and 1 μM TSA by ten passes through a 27-G needle. After centrifugation, the cleared lysates were subjected to immunoprecipitation overnight at 4 °C with anti-HA antibody beads (clone 4B2, Wako Pure Chemical Industries). The beads were incubated with IP buffer containing 5 ng/μL RNase A (Nippon Gene) at room temperature for 15 min, washed five times with IP buffer, and then incubated in IP buffer containing synthesized RNAs (for Fig.8e , 2500 ng Ucp1L RNA; for Supplementary Fig. 6d , 1250 ng Ucp1L RNA + 1250 ng Ucp1C RNA or 1250 ng Ucp1S RNA + 1250 ng Ucp1C RNA). IMP2-bound Ucp1 RNAs were isolated according to the RiboCluster Profiler RIP Assay Kit protocol (MBL) and subjected to real-time qPCR.
HEK293T cells transfected with pcDNA3.1-3×HA, pcDNA3.1-3×HA-IMP2, pcDNA3.1-3×HA-IMP2K438R, or pcDNA3.1-3×HA-IMP2K438Q by the JetPRIME reagent for 24 h were lysed in IP buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM MgCl2, 0.05% NP-40, 1 mM PMSF, and PIC) containing 10 mM nicotinamide and 1 μM TSA by ten passes through a 27-G needle. After centrifugation, the cleared lysates were subjected to immunoprecipitation overnight at 4 °C with anti-HA antibody beads (clone 4B2, Wako Pure Chemical Industries). The beads were incubated with IP buffer containing 5 ng/μL RNase A (Nippon Gene) at room temperature for 15 min, washed five times with IP buffer, and then incubated in IP buffer containing synthesized RNAs (for Fig.
3
Co-immunoprecipitation of PPARγ and SIRT7
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A co‐immunoprecipitation assay was performed as previously described14 . HEK293T cells transfected with the indicated expression plasmids by the jetPRIME reagent for 24 h were lysed in lysis buffer (20 mM Tris‐HCl [pH 7.4], 200 mM NaCl, 2.5 mM MgCl2, 0.5% NP‐40, 1 mM PMSF, protease inhibitor cocktail) containing 10 mM nicotinamide and 1 mM TSA by passing through a 29 G needle (Terumo, Tokyo, Japan) 6 times. After centrifugation at 14,000 g for 10 min at 4°C, cell lysate (1,000 µg) was subjected to immunoprecipitation overnight at 4°C with anti‐HA antibody beads (clone 4B2; Wako Pure Chemical Industries, Ltd.). To detect interactions between endogenous PPARγ and SIRT7, epididymal WAT (epiWAT) was homogenized with a Dounce homogenizer (Tight; ISIS Co., Ltd., Osaka, Japan) in lysis buffer containing 10 mM nicotinamide and 1 mM TSA on ice. After centrifugation at 14,000 g for 10 min at 4°C, cell lysate (1000 µg) was incubated with anti‐PPARγ antibody‐crosslinked resin, which was prepared using a Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific, Rockford, IL), at 4°C overnight for immunoprecipitation. After washing 5 times with lysis buffer, precipitated proteins were eluted with the elution buffer (pH 2.8, containing primary amine) provided in the kit, and detected by western blotting with the respective antibody.
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