How to separate low molecular and high molecular protein from muscle tissue?
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Titin, a giant sarcomeric protein, is involved in the generation of passive tension during muscle contraction, assembly and stability of the sarcomere in striated muscles. Titin gene produces numerous titin protein isoforms with different sizes (∼3–4 MDa) resulting from alternative splicing. To study titin and titin isoform changes under disease conditions, the method to detect and quantify titin protein isoforms is needed. The method reported here is a 1% vertical SDS-agarose gel electrophoresis system that can solubilize, detect and quantify various titin isoform sizes. Sodium dodecyl sulfate (SDS)-agarose gel electrophoresis is an important tool in revealing the size and quantity of giant proteins in the sarcomere. In this method article, heart tissues were dissolved in urea-thiourea-glycerol sample buffer. Muscle proteins were resolved on 1% SDS-agarose gels that were silver-stained subsequently. Titin isoform bands with different sizes were separated on the gel. At the end, we also validated the method for large protein detection. Our results indicated that this electrophoresis method is efficient to study the transitions in titin isoforms.
• This method provides efficient protein extraction with urea-thiourea-glycerol buffer from hard tissues such as striated muscles
• This method provides an efficient way to separate large proteins over 500 kDa
• Combining with silver staining, our method can detect large protein isoforms and quantify the separated protein bands.
Method details
Gel electrophoresis has been used extensively to determine the size and quantity of proteins. Muscle tissues such as cardiac and skeletal commonly express a variety of giant proteins (larger than 0.5 MDa), which play important roles in muscle structure and function. Titin, also called connectin, is the largest known sarcomere protein. In the sarcomere, titin plays a critical role in maintaining structural integrity and developing passive tension with stretch [1]. Titin can thus be viewed as a molecular spring in striated muscle. Titin gene undergoes alternative splicing, and generates numerous isoforms in the heart [2]. The size of different titin isoforms ranges from ∼3 to 4.2 MDa (theoretically 4.2 MDa is full size only when all exons are expressed which has never been detected by SDS-agarose gel so far) in the heart [3]. Titin N2B isoform is produced from a single splicing pathway with a size of approximately 3.0 MDa. N2BA isoforms are produced from multiple splicing pathways, and detectable N2BA isoforms are N2BA-A1 (adult form), A2 (adult form), N1 (embryonic and neonatal form) and N2 (embryonic and neonatal form) with sizes of about 3.4, 3.2, 3.7 and 3.6 MDa respectively [2], [4]. Recently, it has been reported that titin gene splicing is mainly regulated by RNA binding protein 20 (RBM20). In RBM20 knockout rat heart, a new N2BA isoform named N2BA-G with a size of approximately 3.9 MDa has only been expressed [5]. Cardiac titin isoforms alteration has been identified and associated with human heart failure [6], [7]. Gel electrophoresis is the simplest and most direct way to observe the alteration of titin isoforms during developmental and pathological changes. However, electrophoretic analysis of large proteins has been difficult to separate titin isoform proteins. In order to clearly and easily resolve various titin isoforms, SDS-agarose gels have been developed [8]. The present vertical SDS-agarose gel electrophoresis system has been modified and used as an efficient method for high-resolution separation of titin isoforms.
• This method provides efficient protein extraction with urea-thiourea-glycerol buffer from hard tissues such as striated muscles
• This method provides an efficient way to separate large proteins over 500 kDa
• Combining with silver staining, our method can detect large protein isoforms and quantify the separated protein bands.
Method details
Gel electrophoresis has been used extensively to determine the size and quantity of proteins. Muscle tissues such as cardiac and skeletal commonly express a variety of giant proteins (larger than 0.5 MDa), which play important roles in muscle structure and function. Titin, also called connectin, is the largest known sarcomere protein. In the sarcomere, titin plays a critical role in maintaining structural integrity and developing passive tension with stretch [1]. Titin can thus be viewed as a molecular spring in striated muscle. Titin gene undergoes alternative splicing, and generates numerous isoforms in the heart [2]. The size of different titin isoforms ranges from ∼3 to 4.2 MDa (theoretically 4.2 MDa is full size only when all exons are expressed which has never been detected by SDS-agarose gel so far) in the heart [3]. Titin N2B isoform is produced from a single splicing pathway with a size of approximately 3.0 MDa. N2BA isoforms are produced from multiple splicing pathways, and detectable N2BA isoforms are N2BA-A1 (adult form), A2 (adult form), N1 (embryonic and neonatal form) and N2 (embryonic and neonatal form) with sizes of about 3.4, 3.2, 3.7 and 3.6 MDa respectively [2], [4]. Recently, it has been reported that titin gene splicing is mainly regulated by RNA binding protein 20 (RBM20). In RBM20 knockout rat heart, a new N2BA isoform named N2BA-G with a size of approximately 3.9 MDa has only been expressed [5]. Cardiac titin isoforms alteration has been identified and associated with human heart failure [6], [7]. Gel electrophoresis is the simplest and most direct way to observe the alteration of titin isoforms during developmental and pathological changes. However, electrophoretic analysis of large proteins has been difficult to separate titin isoform proteins. In order to clearly and easily resolve various titin isoforms, SDS-agarose gels have been developed [8]. The present vertical SDS-agarose gel electrophoresis system has been modified and used as an efficient method for high-resolution separation of titin isoforms.
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