An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K D ) for a protein/ DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel; the DNA migrates more slowly when bound to a protein. To determine a K D the amount of unbound and protein-bound DNA in the gel is measured as the protein concentration increases. By performing this experiment, students will be introduced to making affinity measurements and gain experience in performing quantitative EMSAs. The experiment describes measuring the K D for the interaction between the chimeric protein GAL4-p53 and its DNA recognition site; however, the techniques are adaptable to other DNA binding proteins. In addition, the basic experiment described can be easily expanded to include additional inquiry-driven experimentation.Keywords: Biochemistry, protein/DNA interaction, equilibrium dissociation constant, EMSA.We present an experiment, appropriate for an undergraduate biochemistry laboratory course, that will enable students to measure the equilibrium dissociation constant (K D ) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs), also known as gel shifts. This experiment introduces students to the theoretical and practical considerations behind measuring the binding affinity for a biological interaction. Equilibrium binding and dissociation constants are typically introduced in general chemistry; however, their application to biological interactions may not be part of biochemistry curricula. Indeed, many biochemistry students, even at the graduate level, do not firmly grasp these concepts. This experiment provides the opportunity to measure the binding affinity of a protein/DNA interaction.The interaction between the protein GAL4-p53 and its DNA recognition sequence is used as a model system. Protein/nucleic acid interactions are fundamental to all of biology, therefore understanding how to quantitatively measure the affinity of such an interaction, and what that affinity means for biological regulation is important. In addition, this experiment will teach students how to perform EMSAs. This is a basic technique that is widely used in biochemical research laboratories, hence is a valuable technique to learn. Lastly, this experiment provides a framework that can be readily expanded, for example, to teach the relationship between K D and rate constants, or to allow for inquiry-driven experimentation.
BACKGROUND AND THEORY
Segmentation of WBMRI in patients with NF1 is feasible and elucidates meaningful relationships among disease phenotype, anthropomorphic and demographic features.
BackgroundCross-sectional imaging is not currently used in planning Total Hip Arthroplasty (THA). The aim of our study is to determine correlations between CT parameters and outcomes following THA.MethodsA prospective registry of patients who underwent total joint arthroplasty was reviewed for patients who: (1) underwent THA, (2) had a CT between 1 year before and 6 months after surgery, and (3) completed perioperative WOMAC and Harris Hip Score (HHS) questionnaires. Two readers measured CT parameters, yielding mean Hounsfield Units, area, average diameter, and perimeter of the psoas major, gluteus medius and minimus muscles. A segmentation algorithm determined visceral and subcutaneous fat area, and waist circumference. ICC was calculated for each measurement to examine inter-reader agreement. Regression analyses were performed to select measurements with most impact on outcome scores.ResultsTwenty-eight patients met inclusion criteria (17 female, 11 male), having mean (+/− standard deviation) age of 54.4 +/− 14.8 years and BMI 29.0 +/− 6.3 kg/m2. Correlations were found between HHS and age (0.650, p = 0.018), height (−1.263, p = 0.009), visceral-to-subcutaneous fat area ratio at the psoas level (0.511, p = 0.018), and waist circumference at the psoas level (1.759, p = 0.002). Inter-reader analysis showed ICC > 0.850 for all measurements.ConclusionAge and height, as well as CT-derived visceral-to-subcutaneous fat area ratio and waist circumference significantly correlate with postsurgical HHS scores following THA. Our study suggests that parameters derived from cross-sectional CT imaging can be useful additional preoperative planning tool for THA.
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