Protein Purification
Introduction
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.
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 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.
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
References: [9] Farrugia A (January 2010). "Albumin usage in clinical medicine: tradition or therapeutic?".Transfus Med Rev 24 (1): 53–63. [12] Campbell and Farrell. Biochemistry, 4th Ed. Brooks/Cole. 2005. Pp. 116-130.