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

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 h...

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Bibliographic Details
Published in:Electrophoresis 2003-06, Vol.24 (11), p.1695-1702
Main Authors: Warren, Chad M., Krzesinski, Paul R., Greaser, Marion L.
Format: Article
Language:English
Subjects:
Agarose gel electrophoresis
Animals
Astacoidea
Blotting, Western
Connectin
Dogs
Electroblotting
Electrophoresis, Agar Gel - methods
Electrophoresis, Agar Gel - standards
Electrophoresis, Polyacrylamide Gel
Humans
Isoforms
Molecular Weight
Muscle Proteins - analysis
Muscle Proteins - isolation & purification
Muscles - chemistry
Protein Isoforms - isolation & purification
Protein Kinases - analysis
Protein Kinases - isolation & purification
Proteins - analysis
Proteins - isolation & purification
Rabbits
Rats
Titin
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Summary: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 ∼13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain (∼ 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.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.200305392