RIP was conducted using the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore) according to the manufacturer’s instruction (Sigma,17701). Briefly, 1 × 106 hiPSC-CMs were resuspended in 50 μl of RIP lysis buffer with protease inhibitor cocktail and RNase inhibitor. For each sample, 5 μg anti-human MYH10 antibody (1:400, Abcam, ab230823) was added and 1 μg isotype IgG antibody (1:200, Cell Signaling, 5415S) was used as the control. RIP–qPCR results were calculated as fold enrichment from specific antibody versus non-specific IgG (1:200, Cell Signaling, 5415S).
Isotype igg antibody
Manufactured by Cell Signaling Technology
Isotype IgG antibody is a type of immunoglobulin G (IgG) antibody that serves as a control for immunoassays. It is designed to have the same isotype as the primary antibody being used, but does not bind to the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ez magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Ab230823, by Abcam (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Pierce bca, by Thermo Fisher Scientific (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Suz12, by Cell Signaling Technology (1 mentions)
Pol2 s2p, by Active Motif (1 mentions)
Pol2 k7m, by Active Motif (1 mentions)
2 protocols using isotype igg antibody
1
RNA Immunoprecipitation of Myosin-10
2
Chromatin Immunoprecipitation Protocol
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Cells were harvested in IP lysis buffer (Thermo Fisher Scientific, 87788), containing protease and phosphatase inhibitors. Lysates were cleared by centrifugation at 10,000 g and protein concentrations were quantified with Pierce BCA (Thermo Fisher Scientific, 23225). Antibodies were coupled to protein A/G magnetic beads (Thermo Fisher Scientific, 26162) for 1 h at 4°C. 500 µg protein lysate was then incubated with 50 μl beads–antibodies complex overnight at 4°C. After repeated washing, proteins were eluted in NuPAGE LDS Sample buffer (Novex, NP0007) at 70°C in 10 min, followed by simple western analysis (Peggy Sue).
Co‐IP was performed using following antibodies: EZH2 (Active Motif, 39933, 1:100), POL2‐S2p (Active Motif, 61083, 1:100), POL2‐K7m (Active Motif, 65691, 1:100) and SUZ12 (Cell Signalling, 3737, 1:100). An isotype IgG antibody (Cell Signaling, 2729S) was used as a negative control.
Co‐IP was performed using following antibodies: EZH2 (Active Motif, 39933, 1:100), POL2‐S2p (Active Motif, 61083, 1:100), POL2‐K7m (Active Motif, 65691, 1:100) and SUZ12 (Cell Signalling, 3737, 1:100). An isotype IgG antibody (Cell Signaling, 2729S) was used as a negative control.
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