Vertical agarose gel electrophoresis and ... - Wiley Online Library

Chad M. Warren Paul R. Krzesinski Marion L. Greaser

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Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

Muscle Biology Laboratory, The electrophoretic separation of high-molecular-weight proteins (.500 kDa) using University of Wisconsin-Madison, polyacrylamide is difficult because gels with a large enough pore size for adequate Madison, WI, USA protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000–4000 kDa) to migrate over 10 cm in a ,13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain (,220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes. Keywords: Agarose gel electrophoresis / Electroblotting / Isoforms / Titin DOI 10.1002/elps.200305392

1 Introduction Gel electrophoresis and immunoblots are useful tools for characterizing proteins. However, using these methods with very large proteins (.500 kDa) is not easy, since protein migration is very limited even in large porosity gels that are mechanically unstable. In addition, transfer efficiency of such proteins out of polyacrylamide gels is low, and this makes Western blotting more challenging. Cardiac and skeletal muscles contain a number of very large proteins. The biggest is titin [1], also described as connectin [2], with a maximum subunit size over 4000 kDa [3]. Several different alternatively spliced isoforms have been described ranging between 700 kDa (novex-3) and 4200 kDa (full length if all exons were expressed) [3]. Another large muscle protein is nebulin with varying sized isoforms (600–900 kDa), and it may function as a molecular ruler to determine thin filament length [4]. Electrophoretic analysis of these very large proteins has been very difficult in the past. Gradient 3.3–12% SDSPAGE was used to separate titin T1(intact titin, Mr ,3300 kDa) and T2 (breakdown product of titin, Mr ,2000 kDa) [5]. A mixture of 2% polyacrylamide and agarose was also used to resolve the large T1 and T2 titin bands [6]. More recently, 2–9.5% acrylamide gradient Correspondence: Dr. Marion L. Greaser, Muscle Biology Laboratory, 1805 Linden Dr. West, Madison, WI 53706, USA E-mail: [email protected] Fax: +608-265-3110 Abbreviations: DATD, N,N’-diallyltartardiamide; VAGE, vertical sodium dodecyl sulfate-agarose gel electrophoresis

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

gels have been utilized to separate the large titin isoforms and fragments [7]. However, acrylamide gradient gels are physically difficult to work with, more complex to pour, and often undergo physical distortion or tearing during fixing and staining. Sequence analyses have revealed several exon splicing patterns as well as species variations in titin expression [7, 8]. The current gel systems cannot clearly and easily resolve the various titin isoforms. SDS-agarose gels have been commercially available from FMC Bioproducts (Rockland, ME, USA) for over 10 years. The ProSieve resolving gel has been used horizontally to separate glycoproteins in a 150–220 kDa range, which was considered high-molecular-weight [9]. Various other types of agarose gels have been used vertically for the separation of proteins in the range of 7–200 kDa [10]. A comparison of agarose and acrylamide gel systems using 22.7 and 86 kDa sized proteins reacted with SDS found better resolution using SeaPrep agarose at up to 6% than with acrylamide [11]. MetaPhorXR agarose has been used in conjunction with SeaKem Gold as the stacking gel to resolve proteins ranging from 6–200 kDa [12]. SDS-agarose gels have been widely used for the separation of proteins, however, there have been no previous published reports using a vertical SDS-agarose gel electrophoresis (VAGE) system for titin characterization. The purpose of the current study was to develop a reliable and reproducible gel electrophoresis system that would more clearly resolve the various titin isoforms. The methods outlined demonstrate high-resolution separation of titin isoforms, and subsequent blot transfer achieves 0173-0835/03/1106–1695 $17.501.50/0

General

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nearly 100% efficiency. This system can also be easily adapted for characterization of other very large proteins from a variety of sources.

2 Materials and methods 2.1 Sample preparation 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 weight of the tissue was determined and sample buffer was added (between 40:1 and 100:1 buffer to tissue v/w ratio). The sample buffer contained 8 M urea, 2 M thiourea, 3% SDS w/v, 75 mM DTT, 0.03% bromophenol blue, and 0.05 M Tris?Cl, pH 6.8 (modified from [13]). Samples were vortexed thoroughly and then left unheated or heated at 1007C for 3 min, 607C for 10 min, or 607C for 20 min. The samples were again vortexed and subsequently centrifuged for 5 min at 13 2006g. The supernatant was removed from the tubes and either immediately loaded on the gels or stored at 2807C.

2.2 Agarose and acrylamide gel preparation Various sizes can be used for the agarose gels, however, 16618 cm gels gave optimum resolution. A Hoefer SE 600 gel unit was used in the current work (Amersham Pharmacia Biotech, Piscataway, NJ, USA), but any vertical gel system with glass plates or plastic disposable gel cassettes will work. The gel plates were thoroughly cleaned and rinsed with 100% ethanol, dried, and assembled using 1.5 mm spacers and placed in the two position gel stand based on the Hoefer SE 600 user manual. A 1 cm high (2.5 mL for our system) acrylamide plug was poured into the bottom of the gel sandwich. The final concentration of the acrylamide plug consisted of 12.8% T acrylamide, 2.34% C N,N’-diallyltartardiamide (DATD) (Bio-Rad, Hercules, CA, USA), [14] 10% v/v glycerol, 0.5 M Tris?Cl, pH 9.3 (Tris-base titrated with HCl), 0.028% w/v ammonium persulfate (APS), and 0.152% v/v N,N,N’,N’-tetramethylethylenediamine (TEMED). The APS was stored as 500 mL aliquots of 10% w/v in a frostfree 2207C freezer. A layer of water was overlayed on the acrylamide plug to facilitate formation of a flat acrylamide interface. The plug was allowed to polymerize for about an hour. Next, the gel unit was inverted, the water layer decanted, and remaining liquid blotted off with paper towels. The stand with the gels was placed into an oven set at 657C and allowed to warm for about 30 min. Also, a

Electrophoresis 2003, 24, 1695–1702 60 cm syringe (luer lock type) and 1.5 mm thick comb (20 lane) were placed in the oven with the gels. The comb had an adjustable back (Amersham Pharmacia Biotech) set for a well depth of 1 cm. It is very important not to exceed 1 cm depth in order to allow clean removal of the comb from the agarose. The final composition of the resolving gel is 1% w/v Sea Kem Gold agarose (Biowhittaker Cell Biology Products, Walkersville, MD), 30% v/v glycerol, 50 mM Tris-base, 0.384 M glycine, and 0.1% w/v SDS (no pH adjustment necessary) (modified from [15]). Agarose powder (0.4 g) was weighed into a 250 mL dry beaker that is 5 times the volume of the resolving gel solution. Then 12 mL of 100% glycerol was pipetted via a 25 mL serological pipet into a clean 50 mL graduated cylinder. Then 8 mL of 56 buffer (0.25 M Tris-base, 1.92 M glycine, and 0.5% SDS, pH 8.5, no pH adjustment necessary) was added and brought up to a final volume of 40 mL with deionized H2O. The graduated cylinder was covered with Parafilm and inverted to mix the solution. When ready to cast the gel, the buffer in the cylinder was poured into the beaker with agarose and swirled to mix. The beaker was covered with plastic wrap and vented with numerous holes and weighed. The beaker was immediately heated in a microwave along with another beaker of deionized H2O (250 mL beaker with 40 mL of water). The agarose was heated until it just began to boil and then swirled gently using an insulated glove. The agarose was heated at least 3 times for a total heating time of about 2 min. The agarose beaker was reweighed and enough of the heated H2O was added back to correct for evaporation loss. The agarose was again swirled, and the mixture placed into the oven for about 5–10 min to allow bubbles to float to the top. The gel was poured in the oven. The prewarmed syringe was used to draw up the agarose solution from the bottom of the beaker; this reduced the amount of bubbles. Then the agarose was injected along the side spacer until the gel plates overflowed with agarose. It was important to fill the syringe with enough agarose to overfill the plates without refilling the syringe (,30 mL). Once the agarose was poured, the gels incubated in the oven for an additional 5 min. The comb was placed in the agarose after which the gel assembly was placed on the bench top for 30 min to solidify the agarose. The gel sandwich was placed in a 47C cold room for at least 30 min, but it could be stored for up to 2 days. The comb was left in the gel until just before sample loading. The 8% acrylamide gel was poured in the same SE 600 gel apparatus as the agarose gel with 0.75 mm spacers. The resolving gel consisted of: 8% T acrylamide, 2.34% C DATD (Bio-Rad) [14], 10% v/v glycerol, 0.5 M Tris?Cl pH 8.8 (Tris-base titrated with HCl), 0.028% w/v ammonium persulfate (APS), and 0.152% v/v TEMED


VAGE and electroblotting of high-molecular-weight proteins

(adapted from [14, 16]). A 3% T acrylamide stacking gel (same as [14]) was poured to allow the protein to migrate 1 cm before entering the resolving gel.

2.3 Electrophoresis Care must be taken not to break the wells during comb removal. The comb should be lifted slightly and rocked perpendicularly to the gel plates until the bottom of the wells break free from the comb. The comb is then removed completely with a smooth slow upward motion. The lower buffer chamber contained 4 L of 50 mM Trisbase, 0.384 M glycine, and 0.1% SDS, and the upper buffer chamber contained 600 mL of the same buffer with the addition of 10 mM 2-mercaptoethanol (modified from [15]). Samples were loaded through the upper buffer, and the gel was run at 15 mA constant current for 5 h. The gels were cooled to 87C with a circulating water bath. Optimum titin separations occurred when the dye front moved to the bottom of the acrylamide plug. When the gel plates were disassembled the acrylamide plug was cut off and discarded. The wells were also cut off at the top to eliminate gel tearing during staining and washing. The acrylamide plug must be removed for the silver staining protocol. The 8% acrylamide gel was run with constant cooling (87C) until the dye front was at the bottom of the gel. The running buffer composition and volumes were the same as for the agarose gels. The gel was run at 16 mA constant current until the dye front had reached the bottom of the gel.

2.4 Staining The silver staining protocol for agarose gels was adapted from one previously described [17]. Polypropylene staining dishes were acid washed with concentrated nitric acid. The gels were fixed in 50% methanol, 12% glacial acetic acid, and 5% w/v glycerol for at least 1 h, but they can be left for up to 2 days in a sealed container. The fixing solution was removed completely, and the gels were dried overnight in a forced air oven set at 377C. (It is important to dry the gels overnight to keep background staining low; the gels may be left in the drier for up to 2 days if needed). All subsequent wash steps were with 500 mL of solution and were performed at room temperature with gentle shaking. The staining solutions were usually made fresh just prior to use to give more consistent staining. The gels were washed with water 3 times for 20 min each. The gels were then washed in potassium ferrocyanide (20 g K4Fe(CN)6 ?3H2O per liter of water) for 5 min and with water 3 times for 5 min each time. The staining solution was prepared by adding solution B to vigorously stirring solution A in equal parts and the

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stain was used immediately. Solution A contains 50 g of sodium carbonate (Na2CO3) in 1 L of water. Solution B contains 2 g ammonium nitrate (NH4NO3), 2 g silver nitrate (AgNO3), 10 g tungstosilicic acid (SiO312WO3 ?26H2O), and 6.7 mL of 37% formaldehyde in 1 L of water mixed in the order given. Solution A is stable for about 2 weeks at room temperature, and solution B is stable for about 5 days protected from light at room temperature. The staining solution was added to the gels and gently shaken until the bands appeared and the background remained low. The staining solution was removed, and the gels were washed with 1% v/v glacial acetic acid for 5 min to stop the staining reaction followed by a water wash for 5 min. Finally the gels were dried between two sheets of mylar with the addition of some glycerol for 2 days on a bench top. The 8% PAGE gels were stained in Coomassie Brilliant Blue R-250 and the agarose gels may also be stained this way. Gels were placed in 0.05% Coomassie Brilliant Blue R-250, 50% v/v methanol, and 10% v/v glacial acetic acid for at least 2 h or overnight. The gels were then destained in 10% v/v methanol and 7.5% v/v glacial acetic acid with the addition of some Kimwipes (to absorb the stain) until the background was nearly clear. This staining protocol was adapted from one previously described [14].

2.5 Electroblotting Immediately after electrophoresis, the agarose gels were placed in transfer buffer (10 mM CAPS, pH 11, 0.1% SDS, 10 mM 2-mercaptoethanol) for equilibration. PVDF membrane was used for all transfers. The transfer was as described [18] except a TE series Transphor unit (Hoefer) was used and the gels were transferred for 2 h and 20 min at 40 V constant voltage. After the transfer, the membrane was stored in 15% methanol.

2.6 Immunostaining The blotting procedure was adapted from one used previously [19]. The transferred titin membranes were blocked with 5% w/v non-fat dry milk in TBST (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.05% Tween 20) overnight at room temperature with shaking. The blots were then rinsed with water 3 times for 10 s each and washed two more times in TBST for 5 min each. The primary antibody H4 (anti-titin monoclonal that binds to titin in the A-band [20]) was diluted to 1 mg/mL in TBST and incubated for 1 hr. Blots were then washed in TBST buffer 6 times for 5 min each before the secondary antibody was applied. The secondary antibody was anti-mouse immunoglobulin G (IgG) (H1L) peroxidase-linked (Amersham Pharmacia Biotech) and was diluted to 1:10 000 with

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TBST and incubated for 1 h. After the secondary antibody incubation, the membranes were washed 6 times in TBST buffer followed by development using the ECL plus kit following manufacturer’s recommendations (Amersham Pharmacia Biotech). The membrane was then exposed to X-OMAT film from Kodak and developed as previously described [19].

2.7 Ferguson and log Mr vs. relative mobility plots Ferguson plots [21] were constructed by plotting the logarithm of the relative mobility of the protein as a function of agarose gel concentration [22]. The gels for the Ferguson plots were run at 300 V constant voltage for 230 min. The relative mobility for the Ferguson and the log molecular weight vs. mobility plots were calculated based on the migration of the lowest-molecular-weight protein in the data set (myosin heavy chain) [22]. The logarithm of the molecular weights of N2A, N2B, I-connectin, and kettin were plotted vs. the relative mobility. The I-connectin (1962 kDa, DDBJ/EMBL/GenBank accession No. BAB 64297.1) and kettin (542 kDa, DDBJ/EMBL/GenBank accession No. BAB64298.1) molecular weights were determined from cDNA sequences previously reported from crayfish claw muscle [23]. The N2A (3816 kDa, DDBJ/EMBL/GenBank accession No. CAD12456) and N2B (2992 kDa, DDBL/EMBL/GenBank accession No. NP_003310) titin isoform molecular weights were also determined from cDNA previously reported from human soleus and human cardiac respectively [24].

3 Results 3.1 Agarose gel electrophoresis Vertical agarose gel electrophoresis (VAGE) overcomes the issues of low protein mobility and small pore size that limit the usefulness of polyacrylamide for very large protein separations. The SeaKem Gold agarose appears to work best for very large proteins. Titin, with an Mr of 3000–4000 kDa, migrated more than 10 cm (Fig. 1) through a ,13 cm resolving gel. The ability of the agarose system to resolve high-molecular-weight proteins allows the demonstration of various titin isoforms in the rabbit that have not been documented as clearly before (Fig. 1). Titin from the rabbit Tibialis anterior appeared to migrate farther into the gel than that from semitendinosus muscle (Fig. 1). The rabbit psoas muscle appears to have two differently sized bands of titin expressed (Fig. 1), which has not been observed previously. Moreover, the rabbit left and right ventricles seemed to have two N2BA type

Figure 1. 1% vertical SDS-agarose gel electrophoresis. RB TA, rabbit tibialis anterior. RB TA1ST, rabbit tibialis anterior and semitendinosus mixed. RB ST, rabbit semitendinosus. DG TS, dog triceps surae (similar to soleus). RT SO, rat soleus. RB SO, rabbit soleus. RB SO1PS, rabbit soleus and psoas mixed. RB PS, rabbit psoas. RB LV, rabbit left ventricle. RB RV, rabbit right ventricle. DG LV, dog left ventricle. The cardiac samples contain N2BA and N2B isoforms whereas the skeletal samples contain only N2A type of titin.

bands (Fig. 1). The triceps surae (similar to soleus) of the dog, rabbit soleus, and rat soleus all differ in size with the biggest difference between the rat and dog (Fig. 1).

3.2 Effect of temperature and heating times on sample preparation The same sample buffer (urea/thiourea, modified from [13]) was used throughout all comparisons. The temperatures and heating times were changed to assess the amount of titin breakdown. The temperatures and times were: 1007C for 3 min with protease inhibitors (Sigma mammalian cocktail 1:100 dilution in sample buffer), 1007C for 3 min, 607C for 20 min, 607C for 10 min, 237C (no heat) with inhibitors, and 237C (no heat, Fig. 2). The



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Figure 2. Agarose gel sample treatments. Inhibitors were Sigma mammalian cocktail 1:100 dilution in sample buffer. Time is in minutes and the temperature was in 7C. The concentrations of the samples in all lanes were the same and were from dog left ventricle. The samples were loaded in two separate concentrations after dilution with sample buffer (1:4 and 1:1).

major titin N2BA (upper) and N2B (lower) isoform bands were clearly resolved under all sample preparation protocols. Samples heated at 1007C had less intact titin and more breakdown products (smear of staining extending to near the bottom of the gel) than those heated at 607C (Fig. 2). The 10 min vs. 20 min incubation time at 607C did not seem to make much difference (Fig. 2). The no heat treatment did not fully dissociate the titin complex and fully solubilize the protein due to apparent protein aggregates that were observed on gels that were overloaded (Fig. 2). The ratios of N2BA to N2B cardiac titin did not appear to significantly change even with varying degrees of breakdown (data not shown). Thus, the 607C heating regime appeared to be optimal, in agreement with previously reported results using skeletal muscle samples [5].

3.3 Transfer efficiency Titin and other high-molecular-weight proteins have not been completely transferred out of acrylamide gels and remain difficult to transfer [14]. Acrylamide gels pre- and post-transfer are shown in Fig. 3b demonstrating virtually complete transfer of myosin heavy chain but only a small proportion of the titin was removed from the gel. Acrylamide gels prepared using the more conventional bisacrylamide cross-linker usually were more difficult to transfer since even the myosin heavy chain often partially remained after transfer. A pre- and a post-transfer agarose gel stained with Coomassie Brilliant Blue R-250 showed no traces of titin remaining after transfer (Fig. 3a). Even silver staining, which is many fold more sensitive [17], still did not show any traces of titin after transfer (Fig. 3c). The Western blot of dog left ventricle with a specific titin antibody demonstrated that the bands seen at this migration distance in the agarose gel were titin (Fig. 3a).

Figure 3. Blotting transfer efficiency. All samples loaded on the gels were dog left ventricle. (A) Coomassie-stained agarose gel pre- and post- transfer. Transferred titin (Western blot) from 1% vertical SDS-agarose gels stained with titin H4 monoclonal antibody. (B) 8% SDS-acrylamide gel, Coomassie-stained before and after electroblotting. The T1 band corresponds to the unresolved N2BA and N2B titin isoforms shown in (A). (C) 1% vertical SDS-agarose gel silver stained pre- and post-electroblotting.

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Figure 5. Ferguson plot based on relative mobility. The logarithm of the relative mobility, mrel is plotted as a function of gel concentration,% agarose. Lines labeled K, I, N2B, and N2A from top to bottom were kettin, I-connectin, N2B, and N2A. The kettin and I-connectin were from crayfish claw muscle. The N2B and N2A titin were from human cardiac left ventricle and human soleus, respectively.

Figure 4. Titin migration differences in 1% vertical SDSagarose gels. DG TS, dog triceps surae (similar to soleus). RT SO, rat soleus. RB SO, rabbit soleus. RB PS, rabbit psoas. The loading was the same for both gels. One gel was run for 150 min and the other for 220 min; both were run at 15 mA constant current.

3.4 Effect of run times The running time for Fig. 1 was optimized for resolution of titin isoforms. Shorter run times allow visualization of the full spectrum of proteins larger than the myosin heavy chain (Fig. 4). Although the resolution for titin was not as good, the pair of psoas titin bands in the rabbit was still observed (Fig. 4). A run time of 2.5 h allowed titin to move approximately 5.25 cm while a run time of about 3.66 h allowed migration of over 7.5 cm (Fig. 4), mobilities unattainable with previous electrophoresis systems. However, proteins with molecular weights smaller than myosin heavy chain (Mr ,220 kDa) cannot be resolved (Fig. 4).

3.5 Plots The Ferguson plot appeared to be linear from 0.75 to 1.5% agarose (Fig. 5). The curve was extrapolated linearly to 0% gel concentration. The assumption was made that

Figure 6. Relationship between the logarithm of molecular weight and relative mobility. The logarithm of the molecular weight, LOG 10 MWt is plotted as a function of relative mobility, mrel. Kettin (542 kDa) and I-connectin (1962 kDa) were from crayfish claw muscle. N2B (2992 kDa) and N2A (3816 kDa) were from human cardiac left ventricle and human soleus, respectively.

if the agarose gel were able to solidify at very low concentrations the relation would remain linear. The straight lines of the Ferguson plot are fairly parallel and do not extrapolate to a common intercept at 0% agarose concentration (Fig. 5). In the log molecular weight vs. relative mobility plot the relation was linear from a molecular weight of ,500 kDa to ,4000 kDa in a 1% agarose gel (Fig. 6).



4 Discussion VAGE can clearly be substituted for SDS-PAGE to separate high-molecular-weight proteins. Major advantages of agarose include increased mobility of very large proteins, improved resolution of isoforms, more mechanical stability of the gels, and much higher transfer efficiency with blots. 1% agarose has far more structural integrity than low percentage acrylamide counterparts. The increased integrity of the agarose is due to both the very high gel strength of the agarose and the increased thickness of the gel (1.5 mm). The main purpose for the acrylamide plug was to keep the agarose resolving gel from sliding out of the plates during electrophoresis. The acrylamide plug included DATD (Bio-Rad) as the cross-linker for the special purpose of increased adherence to the glass plates [16]. This type of cross-linker helped to minimize the amount of acrylamide plug needed and maximized the length of the resolving gel compared to plugs containing the conventional bis-acrylamide cross-linker. Based on the linear plot of log molecular weight vs. relative mobility this gel system appears to separate proteins based on their size (,500 kDa – ,4000 kDa). Since the straight lines of the Ferguson plot are fairly parallel and do not extrapolate to a common intercept the protein mobility decreases with increased molecular weight [22]. Hence, it appears likely that the separate titin bands are true size variants. New variants of titin were observed in rabbit psoas, which clearly has two N2A titin bands. Rabbit cardiac tissue appears to have two N2BA isoforms. In addition, dog triceps surae (similar to soleus), rat soleus, and rabbit soleus N2A titin all have different size variants of N2A titin. The sample heating comparisons showed that the optimal temperature for solubilization of cardiac titin with minimal breakdown was 607C for 10 or 20 min. The titin isoform ratios of the dog cardiac N2BA to N2B did not appear to change significantly even with varying degrees of breakdown (data not shown). This could have important implications when trying to determine titin isoform ratios present due to the inherent variability of protein breakdown. The heating results agree with those previously published on single skeletal muscle fibers [5]. Heating to 607C is important to fully dissociate the titin and break any disulfide bonds before the run. Apparent aggregates of titin were observed on the gel when samples were not heated (Fig. 2). Thus, some heating is important for the complete dissociation and solubilization of the myofibrillar proteins. Inclusion of 2-mercaptoethanol in the upper buffer chamber prevented reformation of disulfide bonds during electrophoresis [14].

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The transfer of the high-molecular-weight proteins from agarose gels approached 100% efficiency. There has been some improvement in the transfer of proteins from acrylamide using a semidry system [25]. However, titin bands were still clearly visible on post transfer Coomassie-stained gels [25]. With the VAGE system there were no visible bands of titin or fragments of titin left on the post transfer gel after silver staining (more sensitive than Coomassie). The increased transfer efficiency is most likely due to the much larger pore size of the agarose compared to typical acrylamide gels. The inclusion of 30% glycerol seemed to help reduce the diffusion of the protein bands. It has been suggested previously [26] that glycerol increases the viscosity and thus reduces the rate of diffusion of the proteins. Glycerol is often used in polyacrylamide gels to improve the resolution of myosin isoforms [27]. An interesting characteristic of the agarose resolving gel was that the dye front (bromophenol blue) migrated near that of the myosin heavy chain but other lower molecular weight proteins migrated ahead of the dye. In conclusion, the vertical agarose gel separation of titin and other high-molecular-weight proteins was simplified and improved compared to previous methods using gradient SDS-PAGE. The transfer efficiency of proteins from agarose gels is nearly 100%. The gel stability is far better than that of acrylamide counterparts. New apparent size variants of titin have been discovered as a result of the improved gel electrophoresis method. The vertical agarose electrophoresis system will also be a useful tool for exploring and characterizing other very large proteins in non-muscle tissues. This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by grants from the National Institutes of Health (HL47053; HL62466). Received October 15, 2002

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