Biochem

Introduction:

Within the many types of experiments conducted in the laboratory, many equations were used, as well as new equations taught. In one of the laboratory periods, the ability to analyze the supernatant samples obtained from the natural preparation method for PL and PG content were taught. When conducting experiments that involve this structure, there are many things to consider when designing assays to evaluate enzyme activity. Within the experiment, is the enzyme gets denatured, it is no longer able to be measured due to the enzyme not functioning because of denature. In the solution, the ionic strength also affects the activity of enzymes. In example, PL or PG may require a small or large amount of a solution in order to be active. Along with new equations and new analysis abilities, the conducting of plasmin assay, plasminogen assay, and conduction of standard curve was taught. Along with PL and PG, another way to examine enzyme reactions is to examine their rate, or velocity. Also, with the new equations and analysis', learning how to use chromomeric enzyme activity assays to evaluate the kinetics of enzyme reactions was taught. One of the general equations used was v=k[P]. This equations was used for the rate of a reaction. Within the laboratory experiments, from analyzing supernatant samples obtained from natural preparation method for PL and PG content, to making and understanding saturation curves were taught, and used in the many experiments conducted in the previous semester.

Materials and Methods:

Supernatant sample isolation:

In order to prepare samples for enzymatic activity assays, 0.75mL of milk and 0.25 mL of 0.5 M of trisodium phosphate were mixed in Eppendorf tubes. Then, the Eppendorf tubes with the solution were shaken by hand for five minutes and incubated at 37(C for 15 minutes. After the tubes were vigorously shaken, the tubes with the solution were centrifuged in a microcentrifuge at 15,600 x g for 15 minutes. After the solutions were taken out of the centrifuge machine, 1.5 mL of the supernatants were obtained from the solution and put in Eppendorf tubes. The pellets in the solution were discarded and the supernatants were stored at -20(C.

β-casein protein assay:

In order to prepare the β-casein standards, about 100µL of supernatant of each sample was added to 5mL of a brown solution. Standard solutions of 0.375, 0.635, 0.875, and 100% (w/v) β-casein were prepared using the β-casein solution and deionized-distilled water (DDW) as shown in Table 1. The solution were put in screw-cap micro-centrifuge tubes and diluted with DDW. A sample β-casein was also prepared to calibrate the spectrophotometer. The absorbance of each sample was measured at A595. The concentrations of the β-casein were already determined by standard curve relating the absorbance to the β-casein concentration sample.

Table 1. β-casein (β-CN) standard solution preparation protocol.

|Standard |% β-CN |1% β-CN solution (µL) |Water (µL) |Samples Buffer (µL) |

|A |0.375 |15 |25 |960 |

|B |0.625 |25 |15 |960 |

|D |0.875 |35 |5 |960 |

|E |1.000 |40 |0 |960 |

In preparing the samples, 5µL of protein sample was mixed with 240µL of sample buffer and put in screw-cap micro-centrifuge tubes. Sample solutions for wach protein sample were made. The tubes with the standards and the samples were shaken by hand for 5 minutes and then boiled for 2 minutes. Then, 15 µL f each sample solution and standard were added to the wells of the Urea Gel according to Figure 1.

Figure 1. Arrangement of where the standards and samples were position in the Urea Gel.

|A |B |D |E |

|1 |0 |25 |75 |

|2 |5 |20 |75