CELL BIO LAB. Biological & Biomedical Sciences Lab Report

Student Name
Professor Name
Course
Date
Cell Biology Lab
Abstract
This paper aims to determine how Triton X-100, a non-denaturing detergent, and centrifugation can be used to distinguish between the globular actin and filamentous actin. Generally, cell disruption protocols are divided into two main categories: mechanical homogenization and temperature treatments and chemical lysis. The mechanical homogenization is all about the use of motorized or handled devices such as rotating blades for breaking down and extracting proteins. Temperature treatments are sometimes used to lyse the cells (Hwang, et al. 112). In this particular technique, the sample is subject to varying cycles of freezing in ethanol bath or dry ice. It is then allowed to stay for some time at 370-degree Celsius; ice crystals are formed in the end, which can be broken down to obtain protein samples. In the method of chemical lysis, the protein sample is suspended in a hypotonic solution using a number of chelating agents, solvents, antibiotics or detergents. It then interacts with specific components of the cell wall and lets proteins leak through the cell wall. This method is primarily used for lysing fungi, yeast and bacteria cells.
Introduction
Cell lysis is the very first step of organelle isolation, protein extraction, and purification as well as cell fractionation. It can be defined as the process of breaking down of the cell membrane through the enzymatic, osmotic or viral mechanism. The fluid that contains the contents of all of the lysed cells is known as a lysate (Grenklo 414). In the fields of biology and chemistry, cell culture has always been the prior choice of researchers when it comes to RNA, extraction, DNA extraction, protein purification and purification of organelles or individual cells. A large number of species of viruses and bacteria are subject to lysis. During this process, if the cell wall is lost entirely, then the bacterium is known as a protoplast. Cell lysis is extensively used in labs to break down and purify open cells and study them for their contents (Theise 337). This experiment aimed to understand how a non-denaturing detergent, centrifugation, and Triton X-100 can be used for separating multiple poles of filamentous actin and globular actin. For this purpose, we first lysed the given cells and observed how the proteins could be separated and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the next step, we had to find out the molecular weight of an unknown sample of proteins. The experiment has been cumulative in nature, and we used all of the techniques we learned during the semester.
Materials
* A T25 flask of CHO cells
* Warm (37°C) trypsin/EDTA solution
* A transfer pipet
* Bleach
* Beakers
* 5 mL of phosphate-buffered saline (PBS)
* The phase contrast microscope
* 10 percent of fetal bovine serum
Method
Part 1: Preparation of the Cell Lysate
* On the first day, the teacher gave us a T25 flask of CHO cells. These cells were cultured in high glucose and supplemented with 10 percent of fetal bovine serum, 2 mM of L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin.
* The culture was observed under the ...