The lysis buffer consisted of 1% Nonidet P‐40, 150 mmol/L NaCl, 20 mmol/L Tris‐HCl (pH 7.5), cOmplete EDTA‐free protease inhibitor cocktail (Roche Life Science) and phosphatase inhibitor cocktail (Nacalai Tesque). For coimmunoprecipitation, 0.5 mL cell lysate was incubated with 25 or 30 μL precoated Dynabeads protein A or M280 sheep antimouse IgG (both from Invitrogen, Thermo Fisher Scientific), respectively, overnight or for 6 hours at 4°C with gentle rotation. Dynabeads were precoated with 2 μg primary antibody per sample. Samples were washed four times with lysis buffer and eluted in SDS‐PAGE loading buffer at 95°C for 3 minutes. Immunoblotting was carried out as previously described.9
M 280 sheep anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in Norway
The M-280 sheep anti-mouse IgG is a laboratory reagent used for the detection and purification of mouse immunoglobulin G (IgG) molecules. It is a suspension of uniform, superparamagnetic polystyrene beads coated with sheep-derived anti-mouse IgG antibodies. This product can be used in various immunoassay and purification techniques that involve the capture and separation of mouse IgG from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Adaptors for paired end sequencing, by Illumina (1 mentions)
Nebnext dna library prep master mix kit, by New England Biolabs (1 mentions)
Masscleave kit, by Labcorp (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
3 protocols using m 280 sheep anti mouse igg
1
Immunoprecipitation and Western Blotting
2
Immunoprecipitation of Membrane Proteins
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Appropriate monoclonal antibody was incubated with 60 µL washed magnetic beads (Dynabeads M-280), coated with M-280 sheep anti-mouse IgG (Invitrogen Dynal AS, Oslo, Norway) for overnight at 4 °C on a rotator (VWR International, Radnor, PA, USA). As negative controls, the coated beads were incubated with either mouse IgG1κ (MOPC21, Sigma) for mAb, or with rabbit γ-globulin (Jackson ImmunoResearch, West Grove, PA, USA) for pAb raised in rabbits. The beads with attached antibody were washed (twice, 200 μL) with phosphate-buffered saline (PBS). Proteins were immunoprecipitated from 1 mg of detergent-extracted total protein by incubation for 4 h at 4 °C with antibody-bound beads. Bead complexes were washed with (four times 200 μL) PTA solution (145 mmol/L NaCl, 10 mmol/L NaH2PO4, 10 mmol/L sodium azide, and 0.5% Tween 20, pH 7.0). Immunoprecipitated proteins were then extracted with 60 μL of 2× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and boiled for 5 min. Following antibodies were used for immunoprecipitation: mouse monoclonal antibody to NCX1 (R3F1, Swant), rabbit polyclonal antibody to NCX1 (p11-13, Swant), mouse monoclonal antibody M75 to CA IX [5 (link)].
3
Genome-wide DNA Methylation Analysis
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Genome-wide DNA methylation profiles of 4F-MEFs, 4F-iTSCs, and TS-EGFP cells were analyzed by meDIP-seq as described previously (Senner et al., 2012) . Briefly, purified genomic DNA was sonicated to yield 150-600 bp fragments and adaptors for paired-end sequencing (Illumina) were ligated using the NEBNext DNA Library Prep Master Mix Kit (New England Biolabs). Immunoprecipitations (IPs) were carried out in duplicate using 500 ng DNA per sample, 1.25 mg anti-5mC antibody (Eurogentec), and 10 ml Dynabeads coupled with M-280 sheep anti-mouse IgG (Invitrogen). The two IPs were pooled and amplified for 12 cycles with adaptor specific primers with index tags for multiplexing (Quail et al., 2012) , run on a 1% agarose gel and fragments ranging between 300 and 500 bp in size were cut out and purified using the QIAGEN Gel Extraction Kit. Sequencing was carried out on an Illumina HiSeq. Reads were mapped to the mouse genome build NCBIM37 and final data analysis was performed using SeqMonk software. For MassArray analysis of individual promoters, DNA was bisulphite-treated using the QIAGEN EpiTect Kit following manufacturer's instructions. Regions of interest were amplified and PCR products were processed using the MassCLEAVE Kit (Sequenom) for MassArray analysis.
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