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CLOCK Protein Analysis

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The mice will be separated into three test groups (N=99), which will have different conditions to analyze the affects of multiple variables on expression of CLOCK proteins. One of the three test groups will be feed a diet that is high in number of lipids and calories, and through the process of in vivo protein transduction, will receive inputs of the CLOCK protein [3]. The purpose of providing both of a high fat diet and an input of the CLOCK protein is to counter effect the potential diminishing effect on expression of CLOCK protein caused by a high fat diet. The second group of mice will have the transient receptor potential channel, TrpV1, via a TrpV1 knockout [4]. It is expected that the lack of TrpV1 will exhibit diminished levels…show more content…
Bimonthly, a random sample of 5 mice will be taken and analyzed for levels of expression of peripheral clock genes in the form of PER proteins in the cytoplasm of cells [5]. The relative levels of the proteins in the cytoplasm will be compared between the three test conditions and analyzed to determine the effects of a high fat diet on the sleep cycle. Each sample of mice will be euthanized and a sample of Epididymal adipose tissue will be taken [6]. Proteins and primary RNA transcript will be extracted from adipose tissue via a TRIzol extraction kit following the protocol published by The Institute for Genomic Research [6]. The sample taken from these mice will have varying levels of PER and CLOCK protein. To help further quantify the relative amounts of these proteins in the extracted sample an ELISA assay will be used. [7]. An ELISA assay will require the purified sample, an antibody specific to the CLOCK protein, and a substrate specific to the antibody [7]. The CLOCK specific antibody will react with the relative amounts of protein extracted from the sample, which, by the addition of a substrate, will be translated into a measurable signal. Once completed, the signal will be analyzed by a plate reader, which uses a program to measure the intensities between the various samples and quantifies the
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