Protein purification is the series of processes to isolate a single type of protein from a complex mixture. This is vital to extract and characterize the protein of interest. However, before doing so, it is important to release the protein from the subcellular organelles. This step is also known as homogenization. This step can be done with the use of blender. As the solution was homogenized, it may undergo saltation or acidation to remove impurities such as calcium anions. Hexane may also be used to defat the protein. Lastly, the solution undergoes differential centrifugation. This will separate the protein crude from the liquid. (Campbell)
After protein purification, the crude now undergoes characterization. Activity assay, Bradford assay, and Warburg-Christian method can be used to characterize proteins. Spectrophotometric analysis are usually used to determine some properties of the protein such as protein concentration.
Results Discussion
A. Invertase from yeast
The prepared yeast is first washed with hexane to defat the protein. After it, the yeast is grinded for 10-15 minutes and is later centrifuged. When the supernatant is clear, it is put in a pre-cooled beaker to prevent denaturation of protein because temperature affects the structure of the protein. Also, ethanol is added to remove some impurities because ethanol here acts as precipitating agent. Later on, the solution is once again centrifuged. The crude is weighed and is washed with acetone to fasten drying. It is then prepared for characterization.
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What causes moldy bread and foods? How can you stop it? If you understand what fermentation is and how yeast survive you can answer these questions. Fermentation is the conversion of pyr uvic acid into other compounds such as lactic acid or ethyl alcohol. Fermentation occurs when cells do not have oxygen. Some Organisms that use fermentation are fungi and bacteria. Yeast are a type of fungus that ...
B. Albumin from egg
Egg white is acquired because it is where albumin is present. It is gently stirred to prevent denaturation and to mix the enzymes present in it. Later on, it is added with 1.0 M HOAc to remove calcium anions present. Then, it is sent to the centrifuge. The precipitate collected is discarded because it contains contaminants which aren’t needed for the experiment. Also, according to Campbell, if the protein of interest is not found in the nuclei, the supernatant is discarded. The solution is placed in a pre-cooled 250 mL E-flask. Pre cooling is important because it prevents denaturation of protein.
Ammonium sulphate is added to salt the protein. This will alter the solubility of the protein and will lessen impurities. After ice bath, the solution is centrifuged once more. The crude is weighed and air-dried and is prepared for characterization.
C. Casein from milk
First, non-fat milk is greatly considered because fat will be included in casein curd. It is later on treated with HCl to remove calcium cation. After treatment, the solution is placed in the centrifuge. The supernatant is discarded because it contains the contaminants (i.e. calcium cations).
Then, ethanol is placed to wash the curd. Ethanol will lessen impurities as well. After decantation, the crude is washed with acetone to fasten drying. The crude is weighed and prepared for characterization.
D. Preparation and characterization of globulin from squash seeds First, two 250-mL are prepared with 2M NaOH and 2M HCl separately which undergone icebath to lower their temperature. Next, 50 grams of squash seeds, washed with distilled water to minimize impurities, were grinded and added with hexane. Hexane helps to defat the protein. Upon addition of 50 mL of hexane, the squash seeds were mixed through a blender which helps the release of the enzymes from the squash seeds. After mixing, the students air dried the seeds for 30 minutes and 100 mL of water was added to it. Then, the students put it into a hotplate for magnetic stirring for about one hour in room temperature. Maintaining typical room temperature will help prevent denaturation of the proteins. 2M NaOH was added after which adjusted the pH to 9.
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The Effects of Crude Protein on Antler Growth in White Tailed Deer Introduction White tailed deer go through drastic endocrine and physiological changes during the year. Due to this reason, their requirements for nutritional supplements are changing quickly. However, the requirement for crude protein seems to be very important for antler growth and to allow optimal development. The present study ...
Later on, the pH was adjusted to 4.5 through addition of 2M HCl. After ice bath, which would make the temperature more convenient for the protein, the solution was placed in eppendorfs which was later centrifuged. RPM at 10000x wil bring down the mitochondria. After collecting the crude, it was supposed to be washed with acetone to fasten the drying period. The mass of the collected crude was 0.48 grams. After that, the students proceed to characterization of protein via quantification of protein concentration by Warburg-Christian method which eventually uses spectrophotometric analysis. Warburg-Christian method is used because it removes the contaminating nucleic acid from 280 nm. Getting the absorbances of the solutions is helpful in determining the protein present such that, it is able to provide certain characteristics of the amino acids of that protein.
For example, absorbances at 280 nm may indicate presence of tyrosine and tryptophan. As for this test, both 280 nm and 260 nm are used. 20 mL of 1% (w/v) globulin solution was needed, thus the students dissolved 0.48 g of the crude in a 48 mL of distilled water. Spectrophotometric analysis for globulin was properly done at 280 and 260 nm. This acquired absorbances of 2.6281 and 2.7505 respectively. Other data were presented in the results.
On the other hand, Bradford assay was also performed with 12 test tubes prepared according to the table given on the protocol. Each absorbance was read at 595 nm. Bradford assay is used as a protein determination method by binding a specific dye (Coomassie Brilliant Blue G-250 dye) to the protein. Anionic blue form of the dye is capable of binding to the protein and is possible to occur at 595 nm. Thus, 595 nm was used for Bradford assay. The tabulated data for Bradford assay is presented in the results.
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Protein assays are designed to measure the total protein in a solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is able to use it to create a standard curve. If this cannot be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are ...
