**3. Conclusion**

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

52 Electrophoresis - Life Sciences Practical Applications

can occur in a few days at room temperature [50, 88, 91].

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

**Figure 5.** Western blotting of titin in striated muscles of ground squirrel; a modified view from [89]. As primary antibodies, the following were used: Z1/Z2 to N-end of titin molecule located in Z-disc of sarcomere (1, 2); AB5 to the part of titin molecule in A-disk located near the M-line of sarcomere (3,4); (1,4) m. soleus; (2,3) myocardium (left ventricle).

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.

In summary, it should be noted that SDS-PAGE of titin is quite difficult and not a routine procedure. Giant molecular mass and the susceptibility of titin to degrade during preparation significantly complicate the study of this protein by electrophoresis. It is necessary to know the three main rules for a successful study of titin by electrophoresis: (1) use protease inhibitors (leupeptin, E-64, protease inhibitor cocktail); (2) do not heat SDS samples higher than 40–60°C; and (3) judiciously select the type of the gel. Currently, the most suitable gels for analyzing titin are the following: (1) vertical agarose-strengthened 2% polyacrylamide gel [85]; (2) vertical 1% agarose gel [70]; and (3) horizontal agarose-strengthened 1.3% polyacrylamide gel [73].

It should be noted that we have obtained experimental evidence of existence in mammalian striated muscles of higher molecular weight isoforms of titin, named NT. According to our data, the development of pathological processes leads to the destruction of NT titins (**Figure 7**),

**Conflict of interest**

**Author details**

Pushchino, Russia

**References**

Ivan M. Vikhlyantsev1,2\* and Zoya A. Podlubnaya1†

† Zoya Podlubnaya died on March 26, 2018

Biology. 2006;**362**(4):664-681

\*Address all correspondence to: ivanvikhlyantsev@gmail.com

2 Pushchino State Institute of Natural Science, Pushchino, Russia

Ivan M. Vikhlyantsev and Zoya A. Podlubnaya declare that there is no conflicts of interest.

Peculiarities of SDS-PAGE of Titin/Connectin http://dx.doi.org/10.5772/intechopen.75902 55

1 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,

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**Figure 7.** Increased proteolysis of titin and reduction of T1 content in m. Soleus of post-apoplectic patient. Vertical agarose-strengthened 2.3% polyacrylamide gel (8.0 × 10.0 × 0.10 cm) was used to separate titin isoforms. (1) Human m. soleus (control); (2) m. soleus of post-apoplectic patient.

which is accompanied by disorders of sarcomeric structure and impairment of the contractile ability of muscles [84]. In addition to fundamental value, these findings are of great practical value, because the testing of changes in titin content in muscles can be used in medical practice to diagnose pathological processes and evaluate effective approaches to their correction.
