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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Circ Cardiovasc Genet. 2012 Feb 1;5(1):o8–o18. doi: 10.1161/CIRCGENETICS.111.960187

Figure 3.

Figure 3

Validation of ANP by Western blotting as identified by LC-MS/MS. A - ANP identified in NRVM medium and NRVM lysate; amounts of total protein loaded into SDS-PAGE gel were 75 µg and 180 µg, respectively; B - ANP in CSC medium and NRVM medium; amounts of total protein loaded into SDS-PAGE gel were 130 µg and 75 µg, respectively. SDS-PAGE conditions: NuPAGE 4–12% Bis-Tris 1.0 mm thick gel (cat. # NP0321; Invitrogen, CA, USA); MES running buffer; 200 V for 35 minutes [17]. The primary antibody (rabbit anti-rat ANP; Pab A4152-35; US Biological, MA, USA) was used in 1:1,000 dilution and secondary antibody-enzyme conjugate (alkaline phosphatase) was used in 1:10,000 dilution. Recombinant rat ANP (1–28 aa from C-terminus, 3063 Da) was from US Biological (MA, USA; cat. # A4152-05). Detailed explanation is given in the text.