Harvested cells were lysed for 40 min on ice in IP lysis buffer (0.75% CHAPS, 10% (vol/vol) glycerol, 150 mM NaCl, 50mM Tris pH 7.5) freshly supplemented with protease inhibitors. Supernatants were diluted to adjust the CHAPS concentration to 0.375%. Whole cell lysates (3 to 5 mg total protein) were incubated with anti-IgG rabbit (Millipore, N101), anti-IgG mouse (Millipore, N103), anti-DNA-PKcs (Thermo Fisher, PIMA513244 or Invitrogen, MA5-132238), anti-SIRT2 (Abcam, ab67299) or anti-GFP antibody (Abcam; Ab6556). Protein G beads, A agarose beads (Invitrogen), or FLAG conjugated beads (Sigma A2095) were used to immunoprecipitate antibody bound protein. Complexes were washed 4 times with IP washing buffer (0.375% CHAPS, 10% glycerol, 150 mM NaCl, 50 mM Tris pH 7.5) supplemented with protease inhibitors.
Anti igg mouse
Manufactured by Merck Group
Sourced in United States
The Anti-IgG mouse is a laboratory reagent used in various immunoassays and research applications. It is a monoclonal antibody that specifically binds to the Fc region of mouse immunoglobulin G (IgG) antibodies. This product can be used to detect, quantify, or purify mouse IgG molecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti igg, by Merck Group (2 mentions)
Rip lysis buffer kit, by Merck Group (2 mentions)
Human anti ago2, by Merck Group (2 mentions)
Ab67299, by Abcam (1 mentions)
Ab6556, by Abcam (1 mentions)
Pima513244, by Thermo Fisher Scientific (1 mentions)
Anti h3k27ac, by Abcam (1 mentions)
Anti h3k4me3, by Abcam (1 mentions)
Anti h3k27me3, by Abcam (1 mentions)
Anti enos, by Santa Cruz Biotechnology (1 mentions)
Anti cox 1, by Cayman Chemical (1 mentions)
Sc 650, by Santa Cruz Biotechnology (1 mentions)
Anti erk2, by Santa Cruz Biotechnology (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Anti cox 2, by Cayman Chemical (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
5 protocols using anti igg mouse
1
Immunoprecipitation of Protein Complexes
2
ChIP Antibodies for dCTCF and Histone Modifications
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The following antibodies were used for ChIP: dCTCF polyclonal antibodies were generated by New England Peptide by immunizing rabbits with a peptide corresponding to the first 20 aminoacids of dCTCF53 (link). The following antibodies were obtained from commercial sources: anti-H3K4me3 (Abcam #8580), anti-H3K27ac (Abcam #4729), anti-H3K27me3 (Abcam #6002), anti-RNA Pol II pSer2 (Abcam #5095), anti-RNA Pol II pSer5 (Abcam #5408), anti-IgG mouse (Millipore #12-371), and anti-IgG Rabbit (Millipore #12-370).
3
Protein Extraction and Western Blot Analysis
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Proteins from mouse abdominal aorta and mAECs were extracted with cold lysis buffer (50 mM Tris•Cl, pH 7.2, 1% (w/v) Triton X-100, 0.1% (w/v) SDS, 500 mM NaCl and 10 mM MgCl2) supplemented with protease and phosphatase inhibitors (Roche). Polyacrylamide gel-electrophoresis and western blot were performed as described [22] . The following primary antibodies were used at the indicated dilutions: anti--actin 1/2000 (sc-47778), anti ERK-2 1/1000 (sc-1647), anti-iNOS 1/1000 (sc-650), and anti-eNOS 1/1500 (sc-654) from Santa Cruz Biotechnology; anti-COX-1 1/1000 (160109) and anti-COX-2 1/1000 (160112) from Cayman Chemical. Secondary HRP-conjugated antibodies were from Santa Cruz Biotechnologies: anti-IgG-rabbit 1/5000 (sc-2004) and anti-IgG-mouse 1/5000 (sc-2005).
Immunocomplexes were detected by incubation with Luminata Forte Western HRP substrate (Millipore).
Immunocomplexes were detected by incubation with Luminata Forte Western HRP substrate (Millipore).
4
Investigating miR-506 Regulation by RIP
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miR-506 mimics or miRNA was transfected into HCCC-9810 and CCLP1 cells. And the cells were lysed and collected in a RIP lysis buffer kit (Millipore, USA). Human anti-Ago2 (Millipore, USA) or mouse anti-IgG (Millipore, USA) were then conjugated with RNAs magnetic beads. Then the expression levels of purified RNA were determined by qRT-PCR.
5
Luciferase Assay and RNA Immunoprecipitation for miR-506 Interactions
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The sequences containing the wild-type (WT) or mutated (MUT) region DDX5 and linc00473 were synthesized by GenePharma (Shanghai, China) and inserted into a pmirGLO-Report luciferase vector. For the luciferase reporter assay, miR-506 mimics and the respective reporter plasmids were transfected into cells using Lipofectamine 3000 according to the manufacturer's protocol. After 24 hours, the Renilla and Fire y luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
RNA-binding protein immunoprecipitation (RIP) assay miR-506 mimics or miRNA was transfected into HCCC-9810 and CCLP1 cells. And the cells were lysed and collected in a RIP lysis buffer kit (Millipore, USA). Human anti-Ago2 (Millipore, USA) or mouse anti-IgG (Millipore, USA) were then conjugated with RNAs magnetic beads. Then the expression levels of puri ed RNA were determined by qRT-PCR.
The immunohistochemistry (IHC) assay IHC analysis was performed as previously described [16] . For this staining, after heat-induced epitope retrieval, para n-embedded sections were incubated with 3% H
RNA-binding protein immunoprecipitation (RIP) assay miR-506 mimics or miRNA was transfected into HCCC-9810 and CCLP1 cells. And the cells were lysed and collected in a RIP lysis buffer kit (Millipore, USA). Human anti-Ago2 (Millipore, USA) or mouse anti-IgG (Millipore, USA) were then conjugated with RNAs magnetic beads. Then the expression levels of puri ed RNA were determined by qRT-PCR.
The immunohistochemistry (IHC) assay IHC analysis was performed as previously described [16] . For this staining, after heat-induced epitope retrieval, para n-embedded sections were incubated with 3% H
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