Use Of Fractionation To Study Cell Contents Reports Example
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Research has always been the way out for the human race. Trying to get meaning out of every puzzle that life has served us on plate. That has been the endeavors that everyone seems to pursue once given chance to roam free. The answers are the key. Similarly, in science, answers have a lot of weight and are the guidelines of how one will perceive the world around. In Medicine, study of the human body from physiological measurements as small as cells to the entire body. To understand the body cell by cell in a step-by-step approach may just be a step forward towards the understanding of the science behind the perfect working of the body. Therefore, to fight to make these dreams valid, this report endeavors to seek out the fractionation concepts and principles as a valid approach to study cellular components adequately. To probe further for the answers to the thesis, an experiment was carried out and will be utilized for explanations.
Fractionation refers to the process where a given mixture (could liquid, gas, solid, isotope or suspension) of certain quantity is broken down into several fractions following a phase transition. The composition of these fractions varies in accordance with a gradient. This concept is widely applied in branches of both technology and science. In the science arena, we wish to test it out on the study of extracted mammalian cells. Borrowed from this separation process is the concept that plays the key role. However, it is significant to note that in the separation of the mammalian cells, the criteria utilized here is the mass difference between the cells. The procedure will also involve maintaining the pH so as to ensure the survival of the cells for study (Rustad and Ghosh, 2012, p. 205104).
This will mean that some chemicals will have to be handled and precautionary measures will have to be put in place. The chemicals include NaCl (Sodium chloride), TRs, Triton-X-100, sodium dodecyl sulphate and BCA reagent. Primarily, due to the fact that they are all laboratory chemicals precautionary measures have to be executed to make sure that no potential accidents in the lab are present. Precautionary measures first include putting on gloves when handling acidic substances such as the BCA reagent and also while handling the cells. The cells could prove dangerous should they be infested with a potential communicable disease. The Triton-X-100 is also a viscous fluid used in the process of extracting cells and should not prove dangerous. Precaution receives recommendation on a just-in-case basis. The same scenario applies to the TRis. Ignorance should not be under any tolerance as the repercussions are unknown and could range from being non-fatal to fatal.
A protease inhibitor is then added to the buffer solution so as to inhibit the natural functions of the proteins when addition of the cells takes place. The preservation of the samples so that they are safe is mandatory backing up the action that involves storing them on ice. 100ul of the solution is added to the wells of caco-2 cells which are scrapped off from the bottom of the wells. The cell suspension is put on ice for incubation for about thirty minutes which follows a series of inversion of the tube after ten minutes in a bid to re-suspend the sample. The tube will be centrifuged at 13,000 rpm for about five minutes and the supernatant is placed in a clean tube.
The secondary section of the experiment leads to the study of the extracted cells. In this case, the cells are being investigated for the amount of protein that is within the sample. This is where the BCA reagent comes in. the reagent changes in color when it comes into contact with the proteins and the color change ranges from green to purple. Proportional to the amounts of protein amount in the subject cell solution is the change in color observable in the experiment. The extent of color change is measured by utilizing the spectrophotometric techniques. The final result is always the standard curve from which the calculation of the amount of protein can be found.
The procedure involves making solutions up in Eppendorf tubes using Bovine Serum Albumin (BSA). With the help of a Gilson pipette, the standards are loaded in triplicate on a 96 well plate. The working reagent is added; BCA reagent A and BCA reagent B. mixing should be properly done for more accurate results. Incubation follows for thirty minutes at room temperature after which the absorbance is read against the plate reader.
The significance of the use of the triton-x-100 and sodium dodecyl sulphate in the buffer is to enhance the property of the buffer to maintain the pH of the cell solution that is used. These cells, in order for them to survive, their maintenance should be in conditions that are similar to the conditions that the body offers. The pH is especially significant for this to be fruitful. The triton-x-100 is viscous and highly soluble in water at temperatures of about 25 degrees Celsius. This makes it very useful at room temperatures. Its viability to be soluble at room temperature makes it an important factor in the creation of the buffer solution (Behera et al 2007, 184501). The sodium dodecyl sulphate is adopted primarily because of its detergent capabilities. It protects the cells extracted from contamination. Its organic nature makes it a better choice as it will not have a negative effect on the sample (Ghosh et al 2007, 204708).
The centrifuge exercise is an action whose importance cannot be understated. Centrifuging will separate the wanted and unwanted parts of the cell solution. It will separate the cell solution of the buffer and the cells from the other members of the sample solution. This is conducted all in a bid to make sure that in the end the sample being tested on is the cell solution and not the other components. The liquid obtained is the wanted sample whereas the solid substance is a compound of the other materials mixed initially to form the buffer solution. Another significance of value attributed to centrifuging is that it ensures that the sufficient amount of protein is isolated for adequate study. Otherwise, the results that one may end up taking up as the right values may either be under or over the right values. Therefore, centrifuging is to be done at the right speed to yield the best results possible (Radeva, Perabo and Sharom 2005, 4930).
The caco-2 cells have been extensively used as representatives of the human gut for a long time now. The cells have its parental cell line in the human colon where they originate. They are cultured using a process called spontaneous differentiation. This process leads to formation of a single layer of cells which express a number of functional and morphological traits distinct to the mature enterocyte. A variety of caco-2 cell lines are utilized in different laboratories with their basis being what they plan to achieve (Yoon et al 2013, 52).
These cells are used as representative in the experiment above because of their absorbing properties. The experiment above extensively depends on the ability of the cells to absorb so that the rate of absorption can be measured against the concentration of the reagents used. In addition, these cells when cultured exhibit the traits of a mature enterocyte which means that all the functions that the cells in the gut are supposed to perform can be performed by this cell in this state making it the best candidate cells for this experiment (Yoon et al 2013, 52).
The principle of fractionation has been a useful entity throughout the experiment as the cells have been divided into various protein standards in order to provide us with control of the experiment. From the standards, a standard curve features and the concentration is finally found as 0.525 ug/mg. evidenced from the above experiments, it can go without denial that fractionation concepts and principles are viable means for study of cell components owing to the success of the above experiment.
Behera, K, Pandey, M, Porel, M, &Pandey, S 2007, 'Unique role of hydrophilic ionic liquid in modifying properties of aqueous Triton X-100', Journal Of Chemical Physics, 127, 18, p. 184501, Academic Search Premier, EBSCOhost, viewed 1 February 2015.
Ghosh, S, Mondal, S, Sahu, K, & Bhattacharyya, K 2007, 'Ultrafast photoinduced electron transfer from dimethylaniline to coumarin dyes in sodium dodecyl sulfate and triton X-100 micelles', Journal Of Chemical Physics, 126, 20, p. 204708, Academic Search Premier, EBSCOhost, viewed 1 February 2015.
Rustad, M, &Ghosh, K 2012, 'Why and how does native topology dictate the folding speed of a protein?', Journal Of Chemical Physics, 137, 20, p. 205104, Academic Search Premier, EBSCOhost, viewed 1 February 2015.
Radeva, G, Perabo, J, &Sharom, F 2005, 'P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains', FEBS Journal, 272, 19, pp. 4924-4937, Academic Search Premier, EBSCOhost, viewed 1 February 2015.
Yoon, H, Ju, J, Kim, H, Park, H, Ji, Y, Lee, J, Shin, H, Do, M, &Holzapfel, W 2013, 'Reduction in cholesterol absorption in Caco-2 cells through the down-regulation of Niemann-Pick C1-like 1 by the putative probiotic strains Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 from fermented foods', International Journal Of Food Sciences & Nutrition, 64, 1, pp. 44-52, Academic Search Premier, EBSCOhost, viewed 1 February 2015.
Taha, M, & Lee, M 2013, 'TES buffer-induced phase separation of aqueous solutions of several water-miscible organic solvents at 298.15 K: Phase diagrams and molecular dynamic simulations', Journal Of Chemical Physics, 138, 24, p. 244501, Academic Search Premier, EBSCOhost, viewed 1 February 2015.
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