Before long, I realized that my goals were unrealistic for the two months I’d be spending at the lab. I saw that I couldn’t build an entire database on huntingtin antibodies in one summer because there are hundreds that have not yet been properly described, and information is often scarce. I did manage to gather sufficient data for eight antibodies, which is a good start.
As for the wet lab project, I completed three modest experiments, each with its own purpose and goal, on the path to determining which of the batches of primary antibodies was better suited to seeing differences between the mutant and wild-type striatal cells. Although I was a bit disappointed that I couldn’t see my project out to its end, I did get a good feel for bench work and for the extensive planning that goes into each experiment.
The first of my small experiments was a primary antibody dilutions test. Dilution is the process of making something weaker or less concentrated. To get different dilutions of the antibody, I added the same amount of antibody to increasingly large amounts of the diluting solution. Then, I applied the dilutions to the mutant and wild-type cells to determine the optimal dilution for seeing the greatest differences between the two types of cells under the confocal microscope. As the dilutions increased, I expected the strength of staining to decrease more rapidly in the wild-type than in the mutant. However, I saw the exact opposite. See below for pictures of the wild-type and mutant cells at the highest and lowest dilutions. Notice the difference in strength of staining between the two.
Fig. 7. Wild-type and mutant cells at lowest and highest dilutions.
Pictures taken at 20x magnification on the confocal microscope. As the dilutions increased, the strength of staining decreased more rapidly for the mutant than the wild-type.
Part of science is dealing with unexpected results. When a scientist initially gets results that seem to go against the hypothesis, he or she must repeat the experiment in order to rule out chance or human error. My second experiment, therefore, was a repeat of the first. Again, I got the same results. Clearly, this was no coincidence.
To look for clues that might explain my results, Surya directed my attention to the method I used to fixate the cells. Using a detergent, I had made tiny holes in the cells’ membranes that allowed the primary and secondary antibodies to flow in. Perhaps I’d used too much detergent and had made the membranes too porous, thereby letting in too much antibody or letting out too many important cellular components. Or there may have been other explanations for the results—maybe the cell types had been mixed up, or maybe the antibody no longer worked. The latter seemed most probable.
My third and final experiment, then, was a test of the amount of detergent, in which I kept the antibody dilutions the same while I varied the concentrations of detergent. On the whole, the results were inconclusive, but Surya plans to run another test in the near future.
Last Modified: 05/22/2009
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