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Electrophoretic Separation of Very Large Molecular Weight Proteins in SDS Agarose

Part of the Methods in Molecular Biology book series (MIMB,volume 1855)


Very large proteins (subunit sizes, >200 kDa) are difficult to electrophoretically separate on polyacrylamide gels. A SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. Proteins with molecular masses between 200 and 4000 kDa can be clearly separated. Inclusion of a reducing agent in the upper reservoir buffer and use of a large pore-sized agarose have been found to be key technical procedures for obtaining optimum protein migration and resolution.

Key words

  • SeaKem Gold agarose
  • Titin
  • DATD
  • Large proteins

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  1. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CrossRef  CAS  Google Scholar 

  2. Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412

    CAS  PubMed  Google Scholar 

  3. Granzier H, Wang K (1993) Gel electrophoresis of giant proteins: solubilization and silver-staining of titin and nebulin from single muscle fiber segments. Electrophoresis 14:56–64

    CrossRef  CAS  Google Scholar 

  4. Tatsumi R, Hattori A (1995) Detection of giant myofibrillar proteins connectin and nebulin by electrophoresis in 2% polyacrylamide slab gels strengthened with agarose. Anal Biochem 224:28–31

    CrossRef  CAS  Google Scholar 

  5. Cazorla O, Freiburg A, Helmes M et al (2000) Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res 86:59–67

    CrossRef  CAS  Google Scholar 

  6. Warren CM, Krzesinski PR, Greaser ML (2003) Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins. Electrophoresis 24:1695–1702

    CrossRef  CAS  Google Scholar 

  7. Razafsky D, Hodzic D (2015) A variant of Nesprin1 giant devoid of KASH domain underlies the molecular etiology of autosomal recessive cerebellar ataxia type I. Neurobiol Dis 15:57–67

    CrossRef  Google Scholar 

  8. Wu JJ, Fujikawa K, McMullen BA et al (2006) Characterization of a core binding site for ADAMTS-13 in the A2 domain of von Willebrand factor. Proc Natl Acad Sci U S A 103:18470–18474

    CrossRef  CAS  Google Scholar 

  9. Ott HW, Griesmacher A, Schnapka-Koepf M et al (2010) Analysis of von Willebrand Factor multimers by simultaneous high- and low-resolution vertical SDS-agarose gel electrophoresis and Cy5-labeled antibody high-sensitivity fluorescence detection. Am J Clin Pathol 133:322–330

    CrossRef  CAS  Google Scholar 

  10. Yamashita K, Yagi H, Hayakawa M et al (2016) Rapid restoration of thrombus formation and high-molecular-weight von Willebrand factor multimers in patients with severe aortic stenosis after valve replacement. J Atheroscler Thromb 23:1150–1158

    CrossRef  Google Scholar 

  11. Tsujii N, Nogami K, Yoshizawa H et al (2016) Influenza-associated thrombotic microangiopathy with unbalanced von Willebrand factor and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 levels in a heterozygous protein S-deficient boy. Pediatr Int 58:926–929

    CrossRef  Google Scholar 

  12. Nishigori N, Matsumoto M, Koyama F et al (2015) von Willebrand factor-rich platelet thrombi in the liver cause sinusoidal obstruction syndrome following oxaliplatin-based chemotherapy. PLoS One 10:e0143136

    CrossRef  Google Scholar 

  13. Hoffner G, Island ML, Djian P (2005) Purification of neuronal inclusions of patients with Huntington’s disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers. J Neurochem 95:125–136

    CrossRef  CAS  Google Scholar 

  14. Radosavljevic J, Nordlund E, Mihajlovic L et al (2014) Sensitizing potential of enzymatically cross-linked peanut proteins in a mouse model of peanut allergy. Mol Nutr Food Res 58:635–646

    CrossRef  CAS  Google Scholar 

  15. Mihajlovic L, Radosavljevic J, Nordlund E et al (2016) Peanut protein structure, polyphenol content and immune response to peanut proteins in vivo are modulated by laccase. Food Funct 7:2357–2366

    CrossRef  CAS  Google Scholar 

  16. Stanic D, Monogioudi E, Dilek E et al (2010) Digestibility and allergenicity assessment of enzymatically crosslinked beta-casein. Mol Nutr Food Res 54:1273–1284

    CrossRef  CAS  Google Scholar 

  17. Oh-Ishi M, Maeda T (2007) Disease proteomics of high-molecular-mass proteins by two-dimensional gel electrophoresis with agarose gels in the first dimension (agarose 2-DE). J Chromat B-Anal Tech Biomed Life Sci 849:211–222

    CrossRef  CAS  Google Scholar 

  18. Yates LD, Greaser ML (1983) Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol 168:123–141

    CrossRef  CAS  Google Scholar 

  19. Fritz JD, Swartz DR, Greaser ML (1989) Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Anal Biochem 180:205–210

    CrossRef  CAS  Google Scholar 

  20. Peats S (1984) Quantitation of protein and DNA in silver-stained agarose gels. Anal Biochem 140:178–182

    CrossRef  CAS  Google Scholar 

  21. Sechi S, Chait BT (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem 70:5150–5158

    CrossRef  CAS  Google Scholar 

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This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and from grants MLG- NIH HL77196 and Hatch NC1131.

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Correspondence to Marion L. Greaser .

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Greaser, M.L., Warren, C.M. (2019). Electrophoretic Separation of Very Large Molecular Weight Proteins in SDS Agarose. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY.

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8792-4

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