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6
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Figure 3. Coomassie brilliant blue staining of protein marker sample
where annatto dye and bromophenol blue (BPB) are compared; a, Annatto
dye alone after staining (without loading protein); b, Annatto dye with
protein marker; c, BPB with protein marker; d, Plant protein sample with
annatto dye; e, Same plant protein sample with BPB; f, Silver nitrate
staining of plant protein sample with annatto extract as the tracking dye; g,
Western blot of GST protein with annatto dye. Proteins in all the lanes
show equal mobility characteristics indicating similarity in the property of
BPB and the dyes used in the present study.
deposited wherever the enzyme is present and appears as a
blue band on the nitrocellulose membrane (Figure 3 g). It
is interesting to compare the chemical properties of bixin
and norbixin with those of BPB. All these compounds are
acidic in nature. However, there are differences in the
overall structure of BPB and these components of the
annatto dye. BPB is aromatic in nature, whereas bixin and
norbixin are unsaturated aliphatic acids. However,
differences in chemical structure do not affect the utility as
a tracking dye in both these cases.
The present investigation advocates the use of natural dyes
instead of BPB as tracking ion front in electrophoretic
techniques. To conclude, we have shown that annatto dye,
a yellow-coloured pigment from the tropical plant B.
orellana L., can be used as a tracking dye for routine SDS–
PAGE of proteins instead of BPB. This procedure is easy,
practical and reliable in case of SDS–PAGE and native
PAGE. Additionally, this dye-yielding plant is commonly
available in tropical regions and is not listed in the
endangered or endemic Red data list 20. The utility of the
extracts from B. orellena as reported in the present study
suggests that there could be many more compounds from
dye-yielding plants that could be used effectively for
similar applications.
1
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CURRENT SCIENCE, VOL. 94, NO. 6, 25 MARCH 2008
12. Hagiwara, A. et al., A thirteen-week oral toxicity study of annatto
extract (norbixin), a natural food color and chemical extracted from
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Additives. WHO Food Additives Series, 1982, vol. 17, pp. 22–27.
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regulatory status of food, drug and cosmetics color additives exempt
from certification. Food Chem. Toxicol., 1995, 33, 515–528.
16. Fernandes, A. C. et al., Norbixin ingestion did not induce any
detectable DNA breakage in liver and kidney but caused a
considerable impairment in plasma glucose levels of rats and mice. J.
Nutr. Biochem., 2002, 13, 411–420.
17. Paumgartten, F., De-Carvalho, R., Araujo, I., Pinto, F., Borges, O.,
Souza, C. and Kuriyama, S., Evaluation of the developmental toxicity
of annatto in the rat. Food Chem. Toxicol., 2002, 40, 1595–1601.
18. Laemmli, U. K., Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature, 1970, 27, 680–685.
19. de Moreno, M. R.., Smith, J. F. and Smith, R. V., Mechanism studies
of coomassie blue and silver staining of proteins. J. Pharm. Sci.,
1986, 75, 907–911.
20. The Wealth of India – A Dictionary of Indian Raw Materials and
Industrial Products, Council of Scientific Research and Industrial
Research, National Institute of Science Communication and
Information Resources, New Delhi, 2003.
ACKNOWLEDGEMENTS. We are grateful to the management of VIT
University for support and encouragement. Special thanks are due to Dr G.
Jayaraman and Dr G. Kalaichelvan for their comments.
Received 29 August 2007; revised accepted 24 January 2008
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