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Protein analysis of Sea Urchin sperm cells

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  1. Introduction.
  2. Results.
  3. Discussion.
  4. Methods.
  5. Conclusion.

Cells express different gene products in order to achieve specialization and differentiation. Sperm are highly specialized to do nothing more than fertilize an oocyte, and they are thus an excellent candidate for research into cellular specialization.1 A terminally differentiated cell like a spermatozoa is useful to study protein composition, because it has a relatively small number of proteins designed to carry out a limited amount of tasks, namely fertilization. Spermatozoa are useful models to study cytoskeleton-dependent motility because their tails, containing the microtubule array necessary for movement, can easily be separated from their heads, which have microtubules for structure and scaffolding rather than motility. Thus, the protein composition of these two sections can make useful comparisons in the study of motility-associated cytoskeleton versus non-motility-associated cytoskeleton.

[...] After purifying sperm head and tail fractions via differential centrifugation, we ran an SDS-PAGE analysis of proteins in these fractions to show two things: first, that western transfer is not 100% efficient, and the dyed gel always has some proteins left behind; second, increasing sample sizes of protein loaded into PAGE wells shows a corresponding increase in band strength. By the sheer thickness and darkness of the bands in the heads of the protein, we can see that proteins are more abundant in the head fractions. [...]

[...] The third phase of our experiment involved western blot (or immunoblot) analysis of ?-tubulin in the sperm fractions. The goal was to find a higher concentration of ?-tubulin in the tail fractions, where it plays the aforementioned role of motility in the sperm flagella. This would not only show that the fractions were homogenized and separated properly using differential centrifugation, but also that ?-tubulin plays a central role in the sperm tails, where it is the main component of the locomotor mechanism, rather than in the sperm head, where it is just one of many cytoskeletal proteins. [...]

[...] According to the data in the western blot assay, some conclusions can be made concerning ?-tubulin abundance in sea urchin sperm cells. There is a higher concentration of ?-tubulin monomers, and thus microtubules, in the tail section of the sperm, suggesting that perhaps the sperm cell has a mechanism of sequestering ?-tubulin monomers in the flagellum for use in association with existing microtubules. We know that microtubules are divided into axonemal (tail) and cytoplasmic (head) classes and that these two classes serve different functions and have different structures. [...]

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