Online Appendix for the following JACC Article
TITLE: Oxidation of Myofibrillar Proteins in Human Heart Failure
AUTHORS: Marcella Canton, PHD,* Sara Menazza, MSC,* Freya L. Sheeran, PHD,†
Patrizia Polverino de Laureto, PHD,‡ Fabio Di Lisa, MD,* Salvatore Pepe, PHD†
Supplemental Methods
Myofibrillar proteins were extracted and subjected to 2D gel electrophoresis followed by
oxyblot analyses. To this aim protein extracts were prepared from human heart tissues as
described (1). Protein samples (120 μg) were resolved by isoelectric focusing on 11-cm
immobilized pH gradient strips (pH 3-10) followed by in-strip DNP derivatization (reacting
with protein carbonyls) as described (2). Second-dimensional separation was performed
by electrophoresis on SDS-PAGE (12% porosity polyacrylamide). After electrophoresis,
the proteins were transferred overnight to nitrocellulose membranes (Bio-Rad) at 150 mA.
The membranes were incubated with anti-DNP antibody, as reported in the Methods
section of the manuscript.
REFERENCES
1. Stanley BA and Van Eyk JE. A solubility optimization protocol for two-dimensional gel
electrophoresis of cardiac tissue. Methods Mol Biol 2007;357:59-65.
2. Conrad CC, Choi J, Malakowsky CA, Talent JM, Dai R, Marshall P, and Gracy RW.
Identification of protein carbonyls after two-dimensional electrophoresis.
Proteomics 2001;1:829-834.
Supplemental Figure 1. Identification of actin and Tm by two-dimensional oxyblot.
Myofibrillar proteins were extracted from NF- and HF-group and subjected to two
dimensional gel electrophoresis followed by detection with anti-DNP antibodies. Red
Ponceau staining was performed for protein loading. The membranes were subsequently
stripped and reprobed with anti-actin and anti-Tm antibodies to confirm the identity of the
carbonylated proteins (indicated by the arrows).