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 α-tubulin in the sperm fractions. The goal was to find a higher concentration of α-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 α-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. [...]
[...] Sample volume Water volume (µg/µl) (µl) (µl) sperm When the gel was finished it was removed from the apparatus and immersed in a dish with 1X western transfer buffer (identical to running buffer except with 20% methanol in place of SDS) for 15 minutes. The filter papers and fiber pads for the western blot were also equilibrated in transfer buffer. The western blot PVDF (polyvinylidene difluoride) membrane was marked to know which side the proteins transfer to. The PVDF membrane was wetted with 20% methanol for 1 minute. [...]
[...] The heads were resuspended in low-pH sea water and examined under the microscope, proving that many sperm were still intact and needed to be rehomogenized; we thus repeated the previous steps once to make our head fraction satisfactorily pure and devoid of tails. Head fraction was spun at 1500g for 5 minutes at 4oC to re-pellet heads; this step was repeated after the resulting pellet was divided into two tubes. These tubes were kept on ice. One tube labeled and one labeled were saved in freezer as back-up samples. [...]
[...] Fraction Concentration (µg/µl) Head 9.61 Tail 0.786 Analysis of the SDS-PAGE gel showed the protein composition in sperm head and tail fractions. Using a Bio-Rad protein ladder, we compared apparent protein bands with known molecular weights. The Coomassie-stained gel is shown in Figure and the mere fact that protein bands appear in this figure shows that the Western Blot transfer is imperfect, leaving some proteins behind on the gel. If the transfer had removed all the proteins from the gel-phase to the solid (membrane) phase, then there would be no banding pattern in Figure which is clearly not the case. [...]
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