To detect titin ubiquitination, LV samples were solubilized in a modified Laemmli buffer (50 mM Tris-HCl at pH 6.8, 8 M urea, 2 M thiourea, 3% SDS w/v, 0.03% ServaBlue w/v, 10% v/v glycerol, 75 mM DTT). For detecting titin oxidation, N-ethylmaleimide instead of DTT was used for solubilization. Samples were heated at 96 °C for 3 min, centrifuged for 3 min at 4 °C at 14,000 rpm, and then separated by agarose-strengthened 2% SDS-PAGE [28 (link),29 (link)]. Gels were run at 2–4 mA constant current per gel for 16 h. Following SDS-PAGE, proteins were blotted onto PVDF membranes (Immobilon-P 0.45 μm; Merck Millipore, Burlington, MA, USA). Blots were preincubated with 4% bovine serum albumin in TBST for 1 h at RT followed by primary antibody incubation overnight at 4 °C.
Titin ubiquitination was investigated by HRP-conjugated secondary antirabbit antibodies (1:10,000), which were added the next day for 1 h at RT. After washing steps, blots were treated with ECL for developing chemiluminescence signal (see above). Chemiluminescence signals were normalized to signals obtained from Coomassie-stained PVDF membranes regarding the total amount of protein transferred. The results were quantified by densitometry using Multi Gauge V3.2 software (Fujifilm Ltd, Tokyo, Kanto, Japan).
Titin ubiquitination was investigated by HRP-conjugated secondary antirabbit antibodies (1:10,000), which were added the next day for 1 h at RT. After washing steps, blots were treated with ECL for developing chemiluminescence signal (see above). Chemiluminescence signals were normalized to signals obtained from Coomassie-stained PVDF membranes regarding the total amount of protein transferred. The results were quantified by densitometry using Multi Gauge V3.2 software (Fujifilm Ltd, Tokyo, Kanto, Japan).
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