Abstract
Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000–3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins.
Key words
- SeaKem agarose
- Titin
- DATD
- Large protein blotting
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Acknowledgements
This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and from grants (MLG-NIH HL77196 and Hatch NC1184).
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Greaser, M.L., Warren, C.M. (2015). Method for Resolution and Western Blotting of Very Large Proteins Using Agarose Electrophoresis. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_30
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DOI: https://doi.org/10.1007/978-1-4939-2694-7_30
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