Anti c myc monoclonal antibody

To identify modifications at the protein level, enriched myofibrillar proteins were isolated from the hearts at 3 months post-birth using F60 buffer (60 mM KCl; 30 mM imidazole; 7.2 mM MgCl2; pH 7.0) with protease/phosphatase inhibitors (Cocktails I and II; Sigma), as described [7 (link)]. Tg protein was confirmed by SDS-PAGE (4–15 % criterion Tris-glycine precast gels; Bio-Rad), followed by Western blots using an anti-c-myc monoclonal antibody (1:1000 dilution, Cell Signaling Tech, Danvers, MA) and anti-cMyBP-C rabbit polyclonal antibody raised against the C0–C1 domains (1: 10,000 dilution). Anti-α-sarcomeric actin monoclonal antibody (1:4000 dilution) is used as a loading control. Detection was carried out using fluorescent-conjugated secondary antibodies in combination with an Odyssey CLx Infrared Imaging System (LI-COR BioSciences, Lincoln, NE).

Paraffin-embedded heart sections were used for immunofluorescence analyses as described [32 (link)]. The following primary antibodies were used: anti-c-myc monoclonal antibody (1:1000 dilution, Cell Signaling Tech), anti-cMyBP-C rabbit polyclonal antibody raised against the C0–C1 domains (1:400 dilution) [6 (link)], and mouse anti-Troponin I (1:1000, Millipore, Billerica, MA). Alexa 488- or Alexa 568-conjugated secondary antibody (Molecular Probes, ThermoFisher, Waltham, MA) directed against mouse or rabbit IgG was used as secondary antibodies, and DAPI (Invitrogen, Carlsbad, CA) was used to identify nuclei.

To prepare whole cell lysates, frozen ventricular tissue derived from the hearts at 3 months post-birth was homogenized in a Bead Beater (Bertin Technologies, Rockvill, MD) in CelLyticM tissue homogenizer buffer (Sigma) freshly supplemented with protease and phosphatase inhibitor cocktails (Roche Applied Sciences) [3 (link)]. The heart extracts were centrifuged at 12,000×g for 15 min and the supernatants collected. Protein lysates were separated on SDS-PAGE using precast 4–15 % Criterion gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad). Membranes were blocked for 1 h in 5 % nonfat dried milk and exposed to primary antibodies overnight. The following primary antibodies were used for immunoblotting: anti-c-myc monoclonal antibody (1:1000 dilution, Cell Signaling Tech), anti-cMyBP-C rabbit polyclonal antibody raised against the C0–C1 domains (1:10,000 dilution), and anti-GAPDH (1:7500 dilution). Detection was carried out using fluorescent-conjugated secondary antibodies (LI-COR) in combination with an Odyssey CLx Infrared Imaging System (LI-COR Biosciences).