Summary
Very large proteins (subunit sizes >200 kDa) are difficult to electrophoretically separate, and they are also challenging to analyze by western blotting because of their incomplete transfer out of polyacrylamide gels. An SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. The large pores of the agarose also allow full transfer of proteins as large as titin (Mr =3,000–3,700 kDa) onto blots. Inclusion of a reducing agent in the upper reservoir buffer and transfer buffer has been found to be a key technical procedure in blotting large proteins.
Key words
- SeaKem agarose
- Titin
- DATD
- Large protein blotting
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Acknowledgments
This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, Purdue University College of Agriculture, and from grants (MLG- NIH HL77196 and Hatch NC1131.
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Greaser, M.L., Warren, C.M. (2009). Efficient Electroblotting of Very Large Proteins Using a Vertical Agarose Electrophoresis System. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_24
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DOI: https://doi.org/10.1007/978-1-59745-542-8_24
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