Table 1.

Types of gels for electrophoretic study of titin

Reference	Type and size of gel, percentage of acrylamide (agarose)	Ratio of acrylamide and bis-acrylamide	Gel buffer, pH	Sample buffer, pH	Heating of samples	Tissue/muscle source	Molecular mass of titin (connectin)	Nuances
1. Wang et al. 1979	3.2% polyacrylamide tube gel, 0.5 × 10 cm	50/1	0.1 M Tris-glycine, pH 8.8, 0.4% SDS	100 mM Tris-HCl, 10 mM EDTA, 40 mM DTT, 10% SDS, 20% glycerol, pH 8.0	Samples were boiled for 2 min	Purified striated muscle myofibrils (chicken breast muscle, rabbit back muscle) prepared according to Etlinger et al. (1976)	Doublet bands (T1 and T2) has Mr ∼1 × 106.	This procedure was a modification of the Etlinger et al. method (1976)
2. Maruyama et al. 1984	1.8–3.0% polyacrylamide tube gel, 0.6 × 10 cm	37/1	7.8 g NaH2PO4 × H2O and 38.6 g Na2HPO4 × 7H20, pH 7.5	0.01 M sodium phosphate buffer, pH 7.0, 1% SDS, and 1% ß-mercaptoethanol, 8 M urea	37 °C for 2 h	Chicken breast muscle	α-connectin – 2.8 MDa; ß-connectin – 2.1 MDa. A faint band above α-connectin is visible in gel (see fig. 2 in Maruyama et al. 1984)	Connectin was extracted from exhaustively washed myofibrils of chicken breast muscle in 0.1 M phosphate buffer, pH 6.6.
3. Hu et al. 1986	1.8% polyacrylamide tube gel, 0.6 × 10 cm	20/1	7.8 g NaH2PO4 × H2O and 38.6 g Na2HPO4 × 7H20, pH 7.5	10% SDS, 40 mM DTT, 10 mM EDTA, 0.1 M Tris-HCl, pH 8.0	2–3 min at 100 °C	Striated muscles of carp, goldfish, bull frog, newt, turtle, snake, chicken, rabbit	Decrease in mobility of α-connectin was in the following order: fish muscles > frog, newt, chicken, snake, rabbit muscles > turtle muscles. As standard, chicken breast muscle was used (MWs of α- and ß-connectins were estimated to be 2.8 and 2.1 × 106, respectively).	This procedure was a modification of the Weber–Osborn method (1969)
4. Wang and Wright 1988	2–12% gradient polyacrylamide slab gel, 8 × 10 cm	50/1	40 mM Tris-acetate, 20 mM Na-acetate, 2 mM EDTA, 0.1% SDS, pH 7.6	1% SDS, 5–10% sucrose, 1 mM EDTA (pH 8.0), 40 mM DTT, 10 pg/ml of pyronin Y (tracking dye), 10 mM Tris-HC1, pH 8.0	90 s at 95 °C	Rabbit striated muscles (longissimus dorsi, psoas, semitendinosus, soleus, diaphragm, adductor magnus, sartorius, heart)	Decrease in mobility of T1 was in the following order: heart > psoas > adductor magnus > longissimus dorsi > semitendinosus, soleus, diaphragm, sartorius.	Muscle tissues were snap-frozen in liquid nitrogen and pulverized to fine powders. Snap-frozen tissue powders were treated with SDS sample buffer preheated to 95 °C. This procedure was a modification of the Somerville and Wang method (1981)
5. Fritz et al. 1989	10% polyacrylamide slab gel (small resolving gel, 6 × 8.2 × 0.075 cm) and 4–10–15% polyacrylamide slab gel (layer resolving gel (12 × 14 × 0.15 cm, each layer 4 cm high).	200/1	0.75 M Tris, 10% glycerol, 0.1% SDS, pH 8.6, 8.8, 9.3	8 M urea, 2 M thiourea, 3% SDS, 75 mM DTT, 25 mM Tris-HCl, pH 6.8.	Samples were boiled for 2–8 min	Myofibrils from rabbit psoas and bovine rectus abdominis muscle	–	Myofibrils were prepared in a buffer containing 50 mM KCl, 20 mM Tris, pH 7.0, 2 mM EDTA, 4 mM MgCl2, 5 mM 2-mercaptoethanol, 0.1 mM PMSF, 1% Triton X-100. Myofibrils were stored at −20 °C in rigor buffer containing 75 mM KCl, 10 mM KH2PO4, 2 mM MgCl2, 2 mM EGTA, 50% (v/v) glycerol, pH 7.0. Migration rates of titin and nebulin in gel are especially affected by 2-mercaptoethanol inclusion in the upper buffer.
6. Wang et al. 1991	2–12% gradient polyacrylamide slab gel, 8 × 10 cm	50/1	40 mM Tris-acetate, 20 mM Na-acetate, 2 mM EDTA, 0.1% SDS, pH 7.6	1% SDS, 5–10% sucrose, 1 mM EDTA (pH 8), 40 mM DTT, 10 pg/ml of pyronin Y (tracking dye), 10 mM Tris-HC1, pH 8	90 s at 95 °C	Rabbit striated muscles (adductor magnus, psoas, longissimus dorsi, sartorius, soleus, semitendinosus, cardiac muscle)	Molecular mass of T1: AM and PS, 2.8 MDa; LD and SA, 2.88 MDa; SO and ST, 2.94 MDa. T2: 2.4 MDa. Cardiac muscle displayed the smallest titin.	Muscle tissues were snap frozen, pulverized in liquid nitrogen, solubilized in hot SDS
7. Horowits 1992	2–12% gradient polyacrylamide slab gel	50/1	40 mM Tris-acetate, 20 mM Na-acetate, 2 mM EDTA, 0.1% SDS, pH 7.6	1% SDS, 10% glycerol, 1 mM EDTA (pH 8), 70 mM ß-mercaptoethanol, 0.05% bromophenol blue, 10 mM Tris-HC1, pH 8	Samples were boiled for 1–2 min	Chemically skinned strips of rabbit psoas and soleus muscles	Soleus titin migrated in gel slightly slower than psoas titin.	Strips of rabbit psoas and soleus muscle were chemically skinned (see the article for more details). After measuring its mechanical properties and dimensions, each single fiber was dissolved in sample buffer.
8. Granzier and Wang 1993	3.3–12% gradient polyacrylamide slab gel, 8 × 10 × 0.075 cm	50/1	40 mM Tris-acetate, 20 mM Na-acetate, 2 mM EDTA, 0.1% SDS, pH 7.6	Laemmli (1970) sample buffer (75 mM Tris-C1, 3% w/v SDS, 120 mM DTT, 55 μg/mL Pyronin Y, 15% v/v glycerol, pH 6.8) was used. Sample buffer of Fritz et al. (1989) (8 M urea, 2 M thiourea, 3% SDS, 75 mM DTT, 25 mM Tris-C1, pH 6.8) was also used.	23–100 °C for 1–8 min. Optimal solubilization temperatures for fibers were in the range of 55–75 °C.	Skinned rabbit psoas fibers.	T1–2.8 MDa; T2–2.4 MDa; A faint band above T1 was detected.	Skinned rabbit psoas fibers were used (see the article for more details). Presoaking in a low ionic strength alkaline buffer was identified as an important step in obtaining complete solubilization of titin from purified myofibrils.
9. Kawamura et al. 1994	2.3–4% polyacrylamide tube gel, 0.6 × 10 cm	36.5/1	0.375 M Tris-HCl, 0.1% SDS, pH 8.8	10% (w/v) SDS, 40 mM DTT, 10 mM EDTA, 0.1 M Tris-HCl buffer, pH 8.0	Samples were boiled for 2 min	Skeletal muscles of lamprey, electric ray, horse mackerel and chicken.	The mobility of ray α- connectin in gel was slower than that of chicken and other animals α-connectins.	SDS gel electrophoresis was carried out according to Laemmli (1970). The electrode buffer (pH 8.3) contained 0.025 M Tris, 0.192 M glycine, 0.1% SDS
10. Granzier and Irving 1995	Slab gel	–	–	50 mM Tris-Cl, 2% SDS, 10% glycerol, 80 mM DTT, 30 μg/ml Pyronin Y, pH 6.8	90 s at 90–95 °C	Rat heart and rabbit skeletal muscles (semitendinosus and psoas)	2.49 ± 0.03 MDa for rat cardiac T1	SDS gel electrophoresis was carried out according to Granzier and Wang (1993). Muscle tissues were quick-froze in liquid nitrogen and pulverized to a fine powder.
11. Tatsumi and Hattori 1995,	2% polyacrylamide slab gel strengthened with agarose (0.5%), 8.3 × 7.2 × 0.15 cm	20/1 (Fairbanks et al. 1971) 37.5/1 (Laemmli 1970)	40 mM Tris-acetate, 20 mM Na-acetate, 2 mM EDTA, 0.1% SDS, pH 7.4 (Fairbanks buffer system) (1971). 0.375 M Tris-HCl, 0.1% SDS, pH 8.6 (Laemmli’s buffer system)	1% SDS, 1% ß-mercaptoethanol, 5 mM EDTA, 5 mM Tris-HC1, pH 8.0, 10% glycerol, 30 μg/ml leupeptin	Samples were boiled for 2 min	Myofibrils of chicken breast muscle (M. pectoralis superficialis) and rabbit skeletal muscles (M. longissimus thoracis, m. soleus)	α- connectin of rabbit muscles had a lower mobility in gel than chicken muscle α- connectin.	Protein samples were frozen at −80 °C until use. Glass cell were cooled for 5 min before pouring of gel. Laemmli’s system provided higher resolution to titin and nebulin.
12. Spierts et al. 1997	2.4–12% gradient polyacrylamide slab gel	–	–	50 mM Tris-Cl, 2% SDS, 10% glycerol, 80 mM DTT, 30 μg/ml Pyronin Y, pH 6.8	90 s at 80 °C	Red and white axial muscles of carp (Cyprinus carpio L.)	Psoas and semitendinosus muscles from rabbit were used as high-molecular-mass standards, and titin of these muscles had a lower mobility than carp muscle T1.	Fiber bundles were quick frozen in liquid nitrogen and pulverized to a fine powder.
13. Cazorla et al. 2000	2–9.5% gradient polyacrylamide slab gel	–	–	–	–	Human soleus, mouse, rat, rabbit, dog, bovine, pig, cow and human myocardium.	Two T1 bands in cardiac muscle and one T1 band in m. soleus were detected. Using the mobility of human soleus T1 (3.7 MDa) and rat cardiac titin T1 (2.97 MDa, bottom band) as standards the authors showed that molecular mass of top cardiac T1 band is ∼3.3 MDa. In addition to T1 and T2 some samples also contained a band that barely entered the gel. Western blotting with titin-specific antibodies indicated that this band is titin. The authors suggested that this is titin aggregates.	Muscle samples were quick-frozen in liquid nitrogen, pulverized, and then rapidly solubilized. SDS gel electrophoresis was carried out according to Granzier and Irving (1995).
14. Warren et al. 2003a, b	1% agarose slab gel, 16 × 18 × 0.15 cm	–	30% v/v glycerol, 50 mM Tris-base, 0.384 M glycine, and 0.1% w/v SDS, pH 8.5 (no pH adjustment necessary).	8 M urea, 2 M thiourea, 3% SDS, 75 mM DTT, 0.03% bromophenol blue, and 0.05 M Tris-Cl, pH 6.8.	100 °C for 3 min, 60 °C for 10–20 min	Rabbit tibialis anterior, semitendinosus, soleus, psoas, left ventricle, right ventricle, dog triceps, left ventricle, rat soleus. The cardiac samples contain N2BA and N2B isoforms whereas the skeletal samples contain only N2A type of titin.	Decrease in mobility of titin was in the following order: cardiac N2B > cardiac N2BA > N2A (rabbit psoas) > N2A (rabbit tibialis anterior, semitendinosus, soleus, rat soleus) > dog triceps. The rabbit psoas muscle has two differently sized N2A bands. The rabbit left and right ventricles have two N2BA bands. Two faint bands above N2B and N2BA were detected. The authors suggested that this is titin aggregates.	Skeletal and cardiac tissue was dissected from New Zealand rabbits (2–3 kg), mongrel dogs, or Sprague Dawley rats and flash frozen in liquid nitrogen. The frozen tissue was pulverized and placed in preweighed Dounce homogenizers. The sample heating comparisons showed that the optimal temperature for solubilization of cardiac titin with minimal breakdown was 60 °C for 10 or 20 min.
15. Neagoe et al. 2003	Agarose-strengthened 2.0% polyacrylamide slab gels with a Laemmli buffer system (Tatsumi and Hattori 1995)	37.5/1	0.375 M Tris-HCl, 0.1% SDS, 0.5% agarose, pH 8.6	1% SDS, 1% 2-mercaptoethanol, 10% glycerol, 8 μg/ml leupeptin, 6 μM bromphenol blue, 4.3 mM Tris–HCl, pH 8.8, 4.3 mM EDTA	Samples were boiled for 3 min	Frog, mouse, hamster, rat, rabbit, cat, cow and human heart, rabbit soleus (freshly excised and frozen muscle tissue).	N2B titin isoform – 3000 MDa; N2BA isoforms – 3.25-3.4 MDa. Rabbit soleus N2A-titin (3.7 MDa) and skeletal nebulin (700–800 kDa) were used as markers for molecular weight detection.	Samples were solubilized by quickly homogenizing 30–60 mg of frozen tissue in 100 μl ice-cold relaxing buffer supplemented with 40 μg/ml leupeptin. Samples were centrifuged briefly and the pellet fraction was used for further analysis.
16. Vikhlyantsev and Podlubnaya 2006	Horizontal 1.3% polyacrylamide gel strengthened with agarose (0.5%), 8.5 × 12.5 × 0.2 cm	37.5/1	0.5 M Tris-HCl, 0.1% SDS, pH 9.0	10 mM Tris–HCl, 1.5% SDS, 1% β -mercaptoethanol, 10% glycerol, 2.5 mM EGTA, 8 μg/ml leupeptin or E64, pH 7.0	Fresh or frozen muscle tissues were incubated in sample buffer for 30–40 min at 20–25 °C (reference samples), after which a small amount of the protein extract was taken for subsequent heating at: 35–60 °C for 10 min, 65–75 °C for 5 min, 80–90 °C for 2–4 min, 95–100 °C for 1–4 min.	Mouse, rat, rabbit, ground squirrel and human striated muscles (freshly excised and frozen muscle tissue).	One or two titin bands (called NT) above N2A, N2BA, N2B and T2 bands were detected. Using the mobility of human and animal nebulin (770–890 kDa) and myosin heavy chain (205 kDa) as standards the authors showed that molecular weights are: 2.08–2.30 MDa (T2), 2.42–2.45 MDa (N2B), 2.56–2.8 MDa (N2A), 3.23–3.3 MDa (cardiac NT), 3.38–3.73 MDA (skeletal NT).	Fresh muscle tissues and frozen in liquid nitrogen were used. Frozen muscles were stored at −80 °C. Recommended temperature for solubilization of titin without breakdown is 35–40 °C.