**2.3. Nuances of sample preparation of titin**

In the paper of Granzier and Wang [88], particular attention was paid to sample preparation of titin. The authors pointed to the fact that titin is extraordinarily sensitive to proteolysis in situ by endogenous proteases and by exogenous proteases such as those found in buffers that are contaminated with bacteria and fungi. According to these and other authors, even in SDSsolubilized myofibril samples, appreciable degradation of titin by residual protease activity can occur in a few days at room temperature [50, 88, 91].

To limit proteolysis, a number of authors suggested the inclusion of protease inhibitors in SDS samples prior to electrophoresis. Leupeptin, E-64, and a protease inhibitor cocktail have been used to inhibit proteolytic degradation of titin [21, 70, 72, 73, 85, 92]. In order to attain better solubilization of titin, it has been proposed to use urea-thiourea SDS DTT sample buffer [70, 93].

Another crucial methodical nuance that should be taken into account during sample`s preparation of titin is heat treatment of the samples. SDS samples are usually prepared by heating them in boiling water for several minutes. This process promotes denaturation of proteins and facilitates disulfide reduction. However, different authors have shown that boiling degrades titin [70, 88, 94, 95]. Samples heated at 100°C had less intact titin and more breakdown products

**Figure 6.** Microphotograph of isolated negatively stained myofibril of rabbit lumbar muscle after the extraction of myosin and actin filaments. The remaining "Z-discs" are kept in the register by longitudinal filaments continually passing through the entire myofibril (indicated by the arrow). Scale bar: 100 nm.

(smears migrating near the bottom of the gel) than those at 60°C [70]. A temperature of 50–60°C for 10–20 min has been considered optimal for the extraction and preservation of intact titin [70, 88, 95]. Results of our studies demonstrated that heating of SDS sample at the said temperature may lead to artifacts in the content of titin. SDS samples of mammalian cardiac muscle heated at 60−65°C for 20 min had another N2BA/N2B ratio than those at 30–40°C [96]. We recommended heating titin in SDS at 35–40°C for 30–40 min [73, 84].
