Whole cell lysates were isolated as described by Hua et al. [4 (link)]. A total of 20 μg of whole cell lysates was loaded onto 10% polyacrylamide gel and separated by Tris/glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer and blocking with Blocking One blocking buffer (nacalai tesque, INC #03953-95), the primary antibodies were incubated with the membrane overnight at 4 °C with gentle rocking, followed by incubation with secondary antibodies for 1 h at room temperature. Finally, proteins were detected using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific; #34075). Antibodies and the dilution of each antibody used for immunoblotting were: anti-PRMT1 (Millipore #07–404; 1:2000), anti-PARP (Cell Signaling #9532S; 1:1000) and β-actin (Sigma #A5441; 1:5000).
Anti prmt1
Manufactured by Merck Group
Anti-PRMT1 is a laboratory product designed to detect and quantify the protein arginine methyltransferase 1 (PRMT1) in biological samples. PRMT1 is an enzyme involved in various cellular processes, including gene expression regulation and signal transduction. The core function of Anti-PRMT1 is to provide researchers with a tool to study PRMT1 expression and activity in their research models.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Anti prmt5, by Merck Group (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti dsrna j2, by Techcomp Instruments (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Anti ph2ax, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Mini protean tetra electrophoresis system, by Bio-Rad (1 mentions)
Anti adma, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Anti mycn, by Santa Cruz Biotechnology (1 mentions)
Anti asym24, by Merck Group (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Immunoblot turbotransfer system, by Bio-Rad (1 mentions)
Ab5823, by Abcam (1 mentions)
4 protocols using anti prmt1
1
Western Blotting Analysis of Protein Expression
2
Western Blot Analysis of Protein Modifications
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Cells and tumor tissues were collected and lysed as described before44 (link). Briefly, 20–100 μg of protein was boiled at 95 °C for 5 min, loaded onto NuPAGE 4–12% Bis-Tris protein gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad) using the Mini-PROTEAN Tetra electrophoresis system (Bio-Rad). Primary antibodies used for membrane staining were anti-PRMT1 (Millipore, 7404; 1:1,000), anti-ADMA (Cell Signaling, 13522S; 1:1,000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233; 1:5,000), anti-pH2A.X (Cell Signaling, 9718S; 1:1,000), anti-β-actin (Santa Cruz Biotechnology, sc-47778; 1:3,000) and anti-dsRNA (J2) (Scicons, 10010200; 1:5,000). Images were collected on an Odyssey scanner (LiCor) or Amershan ImageQuant 80 and analyzed with ImageQuant TL (v8.2.0) and ImageJ (v1.53a).
3
Characterization of PRMT-Mediated MYC Regulation
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Isolation of whole cell lysates and nuclear extracts, immunoblotting, co-immunoprecipitation, purification of baculovirus-infected insect cells and GST fusion proteins, GST pull-down and in vitro methylation assays were performed as previously described [5 (link)]. Antibodies used in this study include anti-PRMT1 (Millipore #07-404), anti-MYCN (Santa Cruz #sc-53993), anti-PRMT5 (Millipore #07-405), anti-β-actin (Sigma #A5316), anti-V5 (Invitrogen #R960-25), anti-phospho-T58 MYC (Applied Biological Material #Y011034), anti-phospho-S62 MYC (BioAcademia #E71-161) and anti-ASYM24 (Millipore #07-414).
4
Quantification and Analysis of PRMT Proteins
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Cells were lysed with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM DTT, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and placed on ice for 30 min. Lysed extracts were centrifuged at 20,913g for 10 min in 4°C. Protein concentration was quantified using Rapid Gold BCA Protein Assay Kit (PIA53227, Thermo Fisher Scientific). Equal amounts of proteins were separated using SDS–PAGE and transferred to nitrocellulose membranes using an immunoblot TurboTransfer system (Bio-Rad). Membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C. The antibodies used are anti-PRMT1 (07-404; Millipore), anti-PRMT5 (07-405; Millipore), anti-H4R3me2a (ab194603; Abcam), anti-H4R3me2s (ab5823; Abcam), and β-actin (A3853; Sigma-Aldrich). Appropriate HRP-conjugated secondary antibodies were applied for 1 h at RT. Proteins were visualized by Western Lightning Plus ECL (PerkinElmer).
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