Experiment 2

Experiment 2 - BC368 Biochemistry of the Cell II Experiment...

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1 BC368 – Biochemistry of the Cell II Experiment 2 A Case Study Experiment: Analysis of Lactate Dehydrogenase Isozymes and Cardiac Troponin I in Blood Samples from ER Patients Introduction Cytosolic proteins are normally retained efficiently inside cells. Tissue damage may compromise membrane integrity sufficiently to allow release of these cytosolic proteins. The insult does not necessarily have to be large enough to result in cell death. Release of tissue proteins into the extracellular environment may occur because of exposure to toxic chemicals, for example in cirrhosis of the liver due to alcohol abuse, or release could be because of some other tissue shock or damage. You and your lab partner are 3 rd year medical students in a coronary care unit. The previous day, there were two admissions of patients with suspected myocardial infarction (heart attack). Both patients were in their forties and had suffered intense chest pains. Patient #1 (male) had been taken ill at a local restaurant, while Patient #2 (female) had just returned home after a strenuous running workout. Two blood samples had been taken from each patient, at approximately 2 hours and 24 hours after the onset of the chest pains, for assay of blood serum lactate dehydrogenase (LDH). Now you are faced with another acute admission (totally unrelated to the first two cases) and there is no available bed in the coronary unit!!! Your problem: Bearing in mind that an initial myocardial infarction may be followed shortly by another, is it possible from the serum LDH data (or from additional data) to decide whether either Patient #1 or Patient #2 could be moved safely to a general medical ward (or even released) to make room in your coronary care unit for the latest admission? What are your deciding factors? Experimental Procedure WEEK 1 – LDH ISOZYME ANALYSIS What would you expect to have happened to blood LDH levels in heart attack patients? During this portion of the project, you will use native gel electrophoresis to separate the different isozymes of LDH that are present in the blood samples of the two patients. By using a specific “activity stain” following this kind of electrophoresis, only the LDH subunits will be displayed, and a very clear interpretation of the makeup of your particular LDH sample can be made. Hopefully it will prove helpful to compare the different LDH’s from the blood samples of your patients. Part 1A Separation of LDH isozymes by charge Differences in charge between proteins will result in differing electrophoretic mobilities, assuming that differences in size do not become important as well. One way to avoid this is to run such charge-separation gels in a matrix with huge pores, such that the size of the protein has no bearing on its mobility. Such is the case when proteins are run through an agarose gel. The agarose gels are preformed for you—they are actually part of a clinical diagnostic kit
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Experiment 2 - BC368 Biochemistry of the Cell II Experiment...

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