Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extractions were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After the incubation with a high affinity anti-METTL3 antibody (1:1000, Abcam, USA), anti-DGCR8 antibody (1:1000, Abcam, USA), anti-PTEN antibody (1:1000, Abcam, USA), anti-β-actin antibody (1:1000, Cell Signaling Technology, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.
Anti dgcr8 antibody
Manufactured by Abcam
Sourced in United States
Anti-DGCR8 antibody is a protein detection tool used to identify the DGCR8 protein in biological samples. DGCR8 is a key component of the microRNA biogenesis pathway. This antibody can be used for applications such as Western blotting, immunoprecipitation, and immunohistochemistry to study the expression and localization of DGCR8.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti pten antibody, by Abcam (1 mentions)
Peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Bca analysis, by Beyotime (1 mentions)
Anti mettl3 antibody, by Abcam (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Anti immunoglobulin g igg, by Merck Group (1 mentions)
Anti m6a antibody, by Cell Signaling Technology (1 mentions)
M6a antibody, by Abcam (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Anti m6a antibody, by Abcam (1 mentions)
Rip immunoprecipitation buffer, by Merck Group (1 mentions)
4 protocols using anti dgcr8 antibody
1
Protein Extraction and Western Blot Analysis
2
DGCR8 and m6A RIP Assay
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The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used for the RIP assay, according to the manufacturer’s instructions. Briefly, stably transfected T24 cells were harvested and lysed in RIP lysis buffer. The RIP lysate was then incubated with magnetic beads conjugated with anti-DGCR8 antibody (Abcam, USA), anti-m6A antibody (Cell Signaling Technology, USA) or anti-immunoglobulin G (IgG, Millipore, USA) antibody at 4 °C overnight. Then, the immunoprecipitated RNAs were extracted using TRIzol reagent (Invitrogen, USA) and subjected to qRT-PCR using primiR-576 primers.
3
RNA Immunoprecipitation and Methylation Analysis
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RNA immunoprecipitation (RIP) assays were conducted using the RNA immunoprecipitation kit (GenSeq, China) in accordance with the manufacturer's instructions. The cells were lysed using RIP lysis buffer and incubated overnight at 4°C with magnetic beads conjugated with anti‐DGCR8 antibody (Abcam, UK) or immunoglobulin G (IgG). Following incubation, the beads underwent treatment with proteinase K and shaking to eliminate any associated protein. The resulting coprecipitated RNAs were then isolated and subjected to RT‐qPCR using pri‐miR‐100 primers, which were subsequently normalized against input.
Methylated RNA immunoprecipitation (MeRIP) was performed using the m6A MeRIP kit (GenSeq, China) as per the manufacturer's instructions. RNAs were fragmented chemically, followed by incubation with magnetic beads conjugated with m6A antibody (Abcam, UK) for immunoprecipitation. The enrichment of m6A‐containing RNA was subsequently analyzed using RT‐qPCR and normalized to input.
Methylated RNA immunoprecipitation (MeRIP) was performed using the m6A MeRIP kit (GenSeq, China) as per the manufacturer's instructions. RNAs were fragmented chemically, followed by incubation with magnetic beads conjugated with m6A antibody (Abcam, UK) for immunoprecipitation. The enrichment of m6A‐containing RNA was subsequently analyzed using RT‐qPCR and normalized to input.
4
METTL3-Mediated m6A Binding Assay
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T24 cells stably transfected with either the METTL3 overexpress lentiviral or control were UV-irradiated at 254 nm, 400 mJ/cm2 (Stratagene Stratalinker), and lysed with RIP lysis buffer (Magna RIP Kit, Millipore, MA) at 4 °C via disruptive sonication. Immunoprecipitations of endogenous DGCR8 were performed using an anti-DGCR8 antibody (1:1000, Abcam, USA) overnight at 4 °C. After washing, the immunoprecipitated protein-RNA complex was analyzed by western blot and treated with Proteinase K. RNAs were extracted by phenol: chloroform: isoamyl alcohol and subjected to qRT-PCR using primers for pri-miRNAs and normalizing to input.
For the m6A RNA binding experiments, the RNAs of T24 cells stably transfected with either the METTL3 overexpress lentiviral or control were isolated and treated with DNase I (Sigma Aldrich, USA). RNAs were fragmented by sonication for 10 s on an ice water mixture. Immunoprecipitations were performed using an anti-m6A antibody (1:1000, Abcam, USA) previously bound to magnetic Dynabeads (Life Technologies, USA) in the RIP Immunoprecipitation buffer (Magna RIP Kit, Millipore, MA) and incubated with DNA-free fragmented RNAs. Beads were then treated with Proteinase K (20 mg/ml) for 1.5 h at 42 °C. RNAs was extracted by phenol: chloroform: isoamyl alcohol and subjected to qRT-PCR using primers for pri-miRNAs and normalizing to input.
For the m6A RNA binding experiments, the RNAs of T24 cells stably transfected with either the METTL3 overexpress lentiviral or control were isolated and treated with DNase I (Sigma Aldrich, USA). RNAs were fragmented by sonication for 10 s on an ice water mixture. Immunoprecipitations were performed using an anti-m6A antibody (1:1000, Abcam, USA) previously bound to magnetic Dynabeads (Life Technologies, USA) in the RIP Immunoprecipitation buffer (Magna RIP Kit, Millipore, MA) and incubated with DNA-free fragmented RNAs. Beads were then treated with Proteinase K (20 mg/ml) for 1.5 h at 42 °C. RNAs was extracted by phenol: chloroform: isoamyl alcohol and subjected to qRT-PCR using primers for pri-miRNAs and normalizing to input.
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