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

Abstract

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate 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 approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 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.

Nuances of electrophoresis study of titin/connectin

“…Using human and animal skeletal muscle myosin heavy chain (205 kDa) and, nebulin (770-890 kDa), as well as the N2A titin isoform (∼3600 kDa and 3700 kDa) of rabbit and human soleus as standards Krüger et al 1991;Granzier and Wang 1993;Prado et al 2005), we estimated that the NT have a Mr of ∼3.8-3.9 × 10 6 (Vikhlyantsev and Podlubnaya 2006). Expression of titin isoforms with these molecular weights is not excluded (Bang et al 2001;Guo et al 2010Guo et al , 2012Li et al 2012), but titin aggregates in gels could not be excluded either (Granzier and Wang 1993;Cazorla et al 2000;Warren et al 2003a). Assuming that molecular masses of titin aggregates should considerably exceed 3800-3900 kDa, we decided to find out more about the differences in electrophoretic mobility of the observed bands.…”

“…With the help of the electrophoresis system using 1% vertical agarose gel (Warren et al 2003a), at least four classes of cardiac N2BA titin isoforms were observed. In particular, two rat embryonic/neonatal forms (N2BA-N1, N2BA-N2) with apparent sizes of approximately 3710 and 3590 kDa, respectively, were found during late embryonic and immediately post-natal (Warren et al 2004).…”

“…To limit proteolysis, the authors suggested the inclusion of protease inhibitors in SDS samples prior to electrophoresis. Leupeptin, E-64, and a protease inhibitor cocktail (Sigma) have been used to inhibit proteolytic degradation of titin (Granzier and Irving 1995;Tatsumi and Hattori 1995;Linke et al 1997;Neagoe et al 2003;Warren et al 2003a;Vikhlyantsev and Podlubnaya 2006). In order to attain better solubilization of titin, it has been proposed to use urea-thiourea SDS DTT sample buffer (Fritz et al 1989;Warren et al 2003a).…”

Titin‐based mechanosensing modulates muscle hypertrophy

“…Samples were centrifuged at 12.000 rpm and the supernatant was divided into smaller aliquots and flash frozen for storage at −80°C. SDS‐agarose (SDS‐AGE) 1% were run on in a Hoefer SE600X vertical gel system (Hoefer Inc; Holliston, USA) was used to electrophoretically separate titin from other proteins 24. Gels were run at 15 mA per gel for 3 h and 15 min, then stained using Neuhoff's Coomassie brilliant blue staining protocol, scanned using a commercial scanner (Epson 800, Epson Corporation, Long Beach CA).…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…Similarly, Biesiadecki et al [76] used a 14% gel with an acrylamide:bisacrylamide ratio of 200:1 to resolve TnT isoforms that differ by as little as three amino acid residues. In order to separate titin isoforms, which can have an MW up to 4 MDa, Warren et al [70,77] developed a vertical SDS-agarose gel system to resolve huge proteins. Compared to SDS-PAGE gels, SDSagarose gels offer improved resolution of titin isoforms, and higher transfer efficiency for subsequent analysis by Western blot.…”