**2.4. Other details of the electrophoretic study of titin**

It is suggested that titin has a tendency to aggregate during electrophoresis, especially in gel systems that use a stacking gel or discontinuous buffers [88]. Fritz et al. [93], as well as Greaser and Warren [97, 98], recommended the inclusion of β-mercaptoethanol in the top anodic buffer to prevent disulfide crosslinking.

There is some peculiarity that should be noted with regard to the preparation of agarosestrengthened 2% polyacrylamide gel. Tatsumi and Hattori [85] to prevent polyacrylamide polymerizing before agarose is polymerized, cooled the glass cell with agarose solution (40°C) for 5 min in ice water. Similarly, we added the agarose solution to glass cells that were pre-cooled to 8–10°C and left the gel for 10 min in the refrigerator at 5°C. Then, we kept the gel for 30 min at 20°C and then for 2–2.5 h at 27°C.

It is recommended to perform electrophoresis using macroporous, agarose-strengthened polyacrylamide gels at low currents. Neagoe (of Wolfgang Linke's group) noted that the best separation of the high molecular weight proteins was obtained by running the electrophoresis overnight at 2 mA per 8.6 × 7.7 cm gel [99]. In our studies with the use of similar gels (8.0 × 10.0 × 0.10 cm), we perform electrophoresis at 3 mA for 40 min, then increasing the current strength up to 7–8 mA [96].

Granzier and Wang recommended to refresh the tank buffer once at 2.5 h to limit pH changes caused by electrolysis during electrophoresis [88].
