BRAFORD ASSAY
Identification of the Marker Enzymes Present in Different Fractions of a Chicken Liver Cell
Priscilla Mariel M. Cadiz
Biology Department, De La Salle University, 2401 Taft Ave, Manila, Philippines
*Email: cadizpriscillamariel@yahoo.com
Cell Fractionation allows the organelles to be studied in more depth and detailed. It is an important technique in Cell Biology because it enables to obtain precise information about the different structure and functions of the organelles. Certain organelles contain specific enzymes with a specific function. These organelles are called Marker Enzymes. In this experiment, four enzymes were assayed namely Alkaline phosphodiesterase, Protease, Peroxidase and Mitochondrial Dehydrogenase. These enzymes were tested in three fractions which were obtained from a chicken liver cell namely the nuclei which contains the nucleus, microsome which contain the mitochondrion and other membrane bound organelles that are part of the endomembrane system and lastly the soluble which consists of the other organelles not mentioned above and different proteins. APDE, peroxidase and mitochondrial dehydrogenase were all incubated at 37 degrees Celsius and color change were noted. The protease on the other hand was subjected to a Differential Scanning Fluorimetry which enables detection of protein stability and its’ binding effects. A Rotor Gene 3000 QPCR Thermocycler was used to obtain the results for the protease activity assay. Results obtained were not ideal and not what was expected. Some enzymes tested positive for the fractions that was expected to be negative and resulted negative for what was supposed to be positive. The fractions that were used might have cause the error of the data and contamination is present.
1. INTRODUCTION
The study of the function of the organelles is an important factor of studying Cell Biology and Biochemistry. Cell Fractionation is the process of studying organelles in much more detail (Chen et al., 2013). Cell
Priscilla Mariel M. Cadiz
Biology Department, De La Salle University, 2401 Taft Ave, Manila, Philippines
*Email: cadizpriscillamariel@yahoo.com
Cell Fractionation allows the organelles to be studied in more depth and detailed. It is an important technique in Cell Biology because it enables to obtain precise information about the different structure and functions of the organelles. Certain organelles contain specific enzymes with a specific function. These organelles are called Marker Enzymes. In this experiment, four enzymes were assayed namely Alkaline phosphodiesterase, Protease, Peroxidase and Mitochondrial Dehydrogenase. These enzymes were tested in three fractions which were obtained from a chicken liver cell namely the nuclei which contains the nucleus, microsome which contain the mitochondrion and other membrane bound organelles that are part of the endomembrane system and lastly the soluble which consists of the other organelles not mentioned above and different proteins. APDE, peroxidase and mitochondrial dehydrogenase were all incubated at 37 degrees Celsius and color change were noted. The protease on the other hand was subjected to a Differential Scanning Fluorimetry which enables detection of protein stability and its’ binding effects. A Rotor Gene 3000 QPCR Thermocycler was used to obtain the results for the protease activity assay. Results obtained were not ideal and not what was expected. Some enzymes tested positive for the fractions that was expected to be negative and resulted negative for what was supposed to be positive. The fractions that were used might have cause the error of the data and contamination is present.
1. INTRODUCTION
The study of the function of the organelles is an important factor of studying Cell Biology and Biochemistry. Cell Fractionation is the process of studying organelles in much more detail (Chen et al., 2013). Cell
References: Chen, Y., Chang, S., Wang, T., Wang, I., & Yong, T. (2013). Cell fractionation on pH-responsive chitosan surface. In Biomaterials (3rd ed., p. 854–863). doi:10.1016/j.biomaterials.2012.10.014 Padh, H Siddiqui, A., & Podesta, R. (1985). Subcellular Fractionation of Hymenolepis diminuta with Special Reference to the Localization of Marker Enzymes. Journal of Parasitology, 71(4), 415-421. Vander Jagt, D., Intress, C., Heidrich, J., Mrema, J., Rieckmann, K., & Heidrich, H. (1982). Marker Enzymes of Plasmodium falciparum and Human Erythrocytes as Indicators of Parasite Purity. Journal of Parasitology, 68(6), 1068-1071.