Electrophoretic charaterisation of Hevein

Yeang H. Y., . and Siti Arija M. A., . and Loke Y. H., . and Mohsin S.M., . Electrophoretic charaterisation of Hevein. pp. 235-244. ISSN 1511-1768

Although the latex protein hevein has received much research interest its analysis by conventional SDS-polyacrylamide gel electrophoresis has been problematic. Hevein does not migrate at a rate that commensurates with its molecular weight and it does not always produce a sharp band on a polyacrylamide gel. To compound this problem hevein is difficult to stain because it has a tendency to diffuse out of the gel during the de-staining process. Moreover hevein Western blot is difficult to perform because as a small protein it easily passes through a nitrocellulose membrane during electro-transfer. Using the tricine SDS-polyacrylamide gel electrophoresis system that is optimised for small proteins and peptides hevein migrated in the gel at a rate consistent with its molecular weight of 4.7 kDa although the migration rate was significantly reduced under non-reducing conditions. Addition of phosphotungstic acid to the Coomassie blue stain fixed hevein and prevented its loss by diffusion out of the gel. Hevein could be effectively Western blotted on to the PVDF membrane and fixed with glutaraldehyde after which the protein was detectable using anti-hevein antibody. Although hevein is an unglycosylated protein it was found to be associated with carbohydrate probably through its lectin property in binding carbohydrate.

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Abstract

Although the latex protein hevein has received much research interest its analysis by conventional SDS-polyacrylamide gel electrophoresis has been problematic. Hevein does not migrate at a rate that commensurates with its molecular weight and it does not always produce a sharp band on a polyacrylamide gel. To compound this problem hevein is difficult to stain because it has a tendency to diffuse out of the gel during the de-staining process. Moreover hevein Western blot is difficult to perform because as a small protein it easily passes through a nitrocellulose membrane during electro-transfer. Using the tricine SDS-polyacrylamide gel electrophoresis system that is optimised for small proteins and peptides hevein migrated in the gel at a rate consistent with its molecular weight of 4.7 kDa although the migration rate was significantly reduced under non-reducing conditions. Addition of phosphotungstic acid to the Coomassie blue stain fixed hevein and prevented its loss by diffusion out of the gel. Hevein could be effectively Western blotted on to the PVDF membrane and fixed with glutaraldehyde after which the protein was detectable using anti-hevein antibody. Although hevein is an unglycosylated protein it was found to be associated with carbohydrate probably through its lectin property in binding carbohydrate.

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Item Type:	Article
AGROVOC Term:	Natural rubber
AGROVOC Term:	Carbohydrates
AGROVOC Term:	Lectins
AGROVOC Term:	Electrophoresis
AGROVOC Term:	Western blotting
AGROVOC Term:	Polyacrylamide
AGROVOC Term:	Polyacrylamide gel electrophoresis
Geographical Term:	Malaysia
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	28 Apr 2025 03:11
URI:	http://webagris.upm.edu.my/id/eprint/23087

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