Non-radioactive method for labelling Trypanosoma brucei brucei (S427, Clone 22) surface proteins using EZ-Link Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6- (Biotinamido) Hexanoate)

  • R. C. Ezeokonkwo
  • W. E. Agu
  • S.J. Black
Keywords: Trypanosoma brucei brucei, Sulfo-NHS-LC-Biotin, Streptavidin-horse radish peroxidase conjugate, SDS-PAGE, Western blot, Molecular weight.

Abstract

Here we describe a non-radioactive procedure that makes use of Ez-linkTM Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6-(Biotinamido) hexanoate to label the surface proteins of Trypanosoma brucei brucei (S427, clone 22). Different concentrations (100ug, 200ug, 400ug, 500ug) of Sulfosuccinimidyl-6-(biotinamido) hexanoate were used for the labelling at different temperatures of 40C, room temperature, 370C. The molecular weights of the labeled surface proteins were determined by running SDS-PAGE, and Western blot. Detection of the biotinylated surface proteins was done by the use of streptavidin-horseradish peroxidase conjugate and exposed with the ECL western blot detection reagent. It was observed that (i) the biological activities of the T.brucei brucei like the motility, viability, and growth were not affected adversely by the biotinylation; (ii) as little as 100ug Sulfo-NHS-LC-Biotin was enough to label about 6x106 Trypanosoma Brucei Brucei; (iii) high temperature appears to have adverse effect on the viability and growth of biotinylated trypanosomas; (iv) three proteins of molecular weights 177.83, 141.25, and 79.43kDa whose actual identities and immunologic importance are not clear at this stage of the study were consistently and clearly labeled by the biotin at different concentrations at room temperature. In comparison with the other alternative methods of labelling surface proteins of parasites described in the literature, this method offers better advantages as there was no radioactive emission; no loss of biological activities like motility, viability, and growth at room temperature and 40C. The method is less cumbersome, and requires less specialized equipment, and more importantly the method is very fast, stable, and reproducible.

(Tropical Veterinarian: 2003 21(1): 14-22)
Published
2004-03-29
Section
Articles

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eISSN: 0794-4845