Introduction
Purification of proteins is an essential technique that provides biochemists with the ability to obtain a sample that contains a specific protein of interest. In order to purify a protein biochemists typically obtain the specific extract, precipitate the protein using ammonium sulfate, preform gel filtration, and lastly run ionic and affinity chromatography. Understanding the use and methods of the different chromatography techniques is of crucial importance in order to successfully purify and conclude the molecular weight of an unknown sample using elution volumes of molecular weight standards.
Gel filtration or exclusion chromatography is preformed to isolate proteins of different sizes between various phases, such as solid or gel and liquid phases. If solid, the gel and beads are denoted as sorbent. Sample mixtures have two phases that they move through, a stationary and mobile phase. The stationary phase is a solid or sorbent, while the mobile phase is gas or water. The process of movement of a sample down a column is referred to as development.
Gel exclusion chromatography is an ideal method used to isolate molecules that are sensitive to harsh environments such as changes in pH or varying concentrations of metal ions. When running gel exclusion chromatography, smaller biomolecules typically penetrate the gel more and thus, take more time to elute through the column. Furthermore, larger biomolecules are too large to penetrate the gel pores and elute through the column at a faster rate. Upon coming out of the Sephedex (or out of the column) biochemists collect fractions, to further the continuation of the experiment. The diagram depicted in the figure below illustrates large molecules and small molecules e...
... middle of paper ...
...h a known molecular weight.
Bibliography
"EXPERIMENTAL TECHNIQUES." Techniques: Chromatography. N.p., n.d. Web. 26 Feb. 2014.
Kumar, Pramod. Ph.D. Experiment 6: Determination of Molecular Weight of a Protein by Gel Exclusion Chromatography. The University of Texas at San Antonio. Pages 1-8.
Moran, Horton et al. Principles of Biochemistry. (2012), page 112.
Selfe, S., Laboratory Manual for Blei and Odian’s General, Organic, and Biochemistry, WH Freeman, New York, 2002, p.195-202.
http://science.csustan.edu/stone/2090/Glucose.htm February 24,2014
Wiggins, Roger C. "Fragmentation and Polymeric Complexes of Albumin in Human Urine." Clinrca Chimica Acta Kelsh Barry.S (1985): 155-63. Online. Web. 24 Feb. 2014. .
To uncover organic compounds like carbohydrates, lipids, proteins and nucleic acid, by using tests like Benedict, Lugol, Biuret and Beta Carotene. Each test was used to determine the presents of different organic molecules in substances. The substances that were tested for in each unknown sample were sugars, starches, fats, and oils. Moreover, carbohydrates are divided into two categories, simple and complex sugars. Additionally, for nonreducing sugars, according to Stanley R. Benedict, the bond is broken only by high heat to make make the molecules have a free aldehydes (Benedict). As for Lipids, there are two categories saturated and unsaturated fats. One of the difference is that saturated fats are mostly solids and have no double bond (Campbell Biology 73). The Beta Carotene test works by dissolving in a lipid, thus giving it color to make it visible. Moreover, proteins are made out of amino acids that are linked by a polypeptide bond (Campbell Biology 75). The purpose of this experiment was to determine whether an unknown class sample or food sample had any carbohydrates, lipids, or proteins in it. The expected result of the lab was that some substances would be present while other would be absent.
Moreover, the sensitivity and specificity of the western blot (Immunoblotting) enables it a common technique for determining specific protein levels in clinical samples. Since the antibody is specific to the antigen immunospecificity it enables the target protein to be identified. Western blotting can produce quantitative data about that protein, which in this case shows the difference between bands in each of the protein samples. The western blot is an analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. The proteins are then transferred to a membrane (in this case, nitrocellulose), where they are stained with antibodies specific to the target protein [1] [2].
The objective of this experiment was to perform extraction. This is a separation and purification technique, based on different solubility of compounds in immiscible solvent mixtures. Extraction is conducted by shaking the solution with the solvent, until two layers are formed. One layer can then be separated from the other. If the separation does not happen in one try, multiple attempts may be needed.
Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
In preparing for the quantitative test for the Bio-Rad protein assay, a spectrophotometer was switched on. Ten test tubes were used and that the known and unknown protein samples were tested duplicate. Tubes one and two were the 0.2 mg/ml protein, three and four 0.3 mg/ml protein, five and six 0.6 mg/ml protein, seven and eight 0.9 mg/ml protein, and lastly nine and ten
Due to the nature of amino acids, a titration curve can be employed to identify
Dr.Grundmann, Oliver, Fundamental of Pharmaceutical chemistry (PHA6432), Module 1, Drug action and drug discovery, Fall 2015
Michener, William K. and Haeuber, Richard A., Bioscience. American Institute of Biological Science. Sep98. Vol. 48. Issue 9. p677.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
Stationary phase is of extreme importance in an HPLC analysis, as the chemical nature of the same and its compatibility with the analyte of interest is extremely significant for efficient separation. The most commonly used stationary phase is silica packed column which acts as a adsorbent. Each component in the sample interacts with these silica particles and gets eluted out in different time intervals. These silica columns may be of C14 or C18 type depending on the component of interest and also the columns themselves come in various dimensions each with a specific purpose of analysis.
Agarose gel electrophoresis separates molecules by to their size, shape, and charge. Biomolecules such as DNA, RNA and proteins, are some examples. Buffered samples such as glycerol and glucose are loaded into a gel. An electrical current is placed across the gel. The current moves the molecules towards the cathode or anode. The speed of the moving molecules depends on the size, shape, and charge. The properties of the gel will definitely affect the movement. Small molecules are expected to move easily and faster thru the pores.
Pauly, S. (2011, February). News from ABC: changes and challenges. Analytical & Bioanalytical Chemistry. pp. 1003-1004. doi:10.1007/s00216-010-4459-0.
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
Materials and Methods: An ion exchange chromatography column was obtained and set up for purification with the addition of 0.5 ml ion exchange matrix. 1 ml