Suzanne Murray G00324885 Group D
Thursday 10 am lab 3/11/16
Introduction
The aim of this experiment is to separate the protein samples based on their molecular size using the SDS-PAGE technique and to detect EGFP protein by carrying out a western blot.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used in the lab for the separation of proteins by their molecular weight. SDS is a detergent used in PAGE because its main role is to break down the disulphide bonds which disrupts the tertiary structure of the proteins making them into a linear shape. It then coats the protein with a negative charge which deactivates the charge that is present on the R groups of the amino acids. Once SDS has carried out their role the charge of the protein is then proportional to its molecular weight. (Oswald et al, 2016)
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It involves transferring or blotting g proteins that were separated using electrophoresis form the gel to a membrane where they can then be seen and examined. It is mainly used to identify if antibodies are present in a protein sample, which can then diagnose many conditions such as HIV and Lyme disease. It is mainly used in hospitals to do so. (The Human Protein Atlas, 2012)
The method used for staining in this lab is the CN/DAB development solution. It contains substrate and peroxide solutions for combined chloronaphthol and diaminobenzidine based detection of horseradish peroxidase (HRP) activity in blotting and tissue staining methods. This solution stains the protein bands a dark turquoise blue which makes them easy to
The transducer in the assay was the Shimadzu UVmini-1240 UV-Vis Spectrophotometer. It is used to measure the absorbance of ferricyanide in solution. Ferricyanide is a yellow species that be measured and compared to the glucose concentration of the sample. Electrochemical glucometers look like the most common type of transducer for commercial use. It utilizes electrodes and flowing current measured by a voltmeter.2
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
Western blot has been a revolutionary technique for identifying the expression of proteins within relative molecular biological samples that shared the same ancestor. 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 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 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].
During this time, it could only be used in a lab with semi-intense supervision. Now, fast forward a few decades and there are D.I.Y. at home kits. The process of Electrophoresis starts with an electric current being run through a gel containing the molecules of interest. The molecules will then travel through the gel in different directions and speeds, based on their size and charge, allowing them to be separated from each other. Dyes, fluorescent tags, and radioactive labels can all enable the molecules on the gel to be seen after they have been separated. Because of these identification markers, they appear as a band across the top of the gel. Electrophoresis can be used for many different things. It is used to identify and study DNA or DNA fragments, and helps us to better understand the molecular components of both living and deceased organisms. Electrophoresis can also be used to test for genes related to specific diseases and life altering diagnoses such as Multiple Sclerosis, Down’s Syndrome, kidney disease, and some types of cancer. Electrophoresis also plays a major role in the testing of antibiotics. It can be used to determine the purity and concentration of one specific type of antibiotic or several general antibiotics at a time. Electrophoresis is also extremely useful in the creation and testing of
Enzymes are proteins that increase the speed of reactions in cells. They are catalysts in these reactions which means that they increase the speed of the reaction without being consumed or changed during the reactions. Cofactors are required by some enzymes to be able to carry out their reactions by obtaining the correct shape to bind to the other molecules of the reaction. Chelating agents are compounds that can disrupt enzyme reactions by binding to metallic ions and change the shape of an enzyme. Catechol is an organic molecule present under the surface of plants. When plants are injured, catechol is exposed to oxygen and benzoquinone is released because of the oxidation of catechol. Catecholase aids in the reaction to produce
Antimicrobial drugs that block protein synthesis react with ribosomal-mRNA complexes. These drugs are safe only because bacterial ribosomes are different in size and structure compared to human ribosomes, however, they can damage human mitochondria since they can contain ribosomes like bacterial ribosomes.
In this experiment the enzyme peroxidase and the substrate hydrogen peroxide were not mixed initially, instead they were both placed in separate tubes and were incubated at a specific temperature, to prevent hydrogen peroxide from undergoing any reaction with peroxidase until they both acquire the required temperature.
We washed off the primary antibody from a membrane at the same manner as we washed off the milk, and then incubated the blot for an hour at room temperature with the secondary antibody GAR680, from this point a membrane was shielded from the light until it has been imaged. The secondary antibody specifically recognizes the primary antibody. GAR680 is Goat Anti-Rabbit immunoglobin antibody. This is an antibody raised in goats that recognizes rabbit immunoglobin. Excess secondary antibody is removed by washing in PBST buffer as was done for the primary antibody, the final wash with 1X PBS to remove residual detergent Tween 20. The membrane was ready for scan and reading on Li-Cor machine. Specially designated tray for fluorescent antibodies was cleaned up with deionized water and kimwipes, then only the membrane was placed on it and entered the Li-Cor machine. The membrane position was from the first to the 10th well, from left to the right, the label was on the left side. The membrane reading was made on 700 nm
Proteins that digested inside of the GI tract consist of not only dietary proteins but also 15-25 grams of enzyme proteins. There are customarily about 125 grams of dietary proteins needed per day. The proteins that are secreted inside of the GI tract lumen by its numerous glands and equal about the amount of protein derived from sloughed and disintegrating mucosal cells. Within healthy individuals, nearly all of this protein is digested all the way to its amino acid monomers.
According to the graph on amylase activity at various enzyme concentration (graph 1), the increase of enzyme dilution results in a slower decrease of amylose percentage. Looking at the graph, the amylose percentage decreases at a fast rate with the undiluted enzyme. However, the enzyme dilution with a concentration of 1:3 decreased at a slow rate over time. Additionally, the higher the enzyme dilution, the higher the amylose percentage. For example, in the graph it can be seen that the enzyme dilution with a 1:9 concentration increased over time. However, there is a drastic increase after four minutes, but this is most likely a result of the error that was encountered during the experiment. The undiluted enzyme and the enzyme dilution had a low amylose percentage because there was high enzyme activity. Also, there was an increase in amylose percentage with the enzyme dilution with a 1: 9 concentrations because there was low enzyme activity.
= 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.
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...
Protein synthesis is one of the most fundamental biological processes. To start off, a protein is made in a ribosome. There are many cellular mechanisms involved with protein synthesis. Before the process of protein synthesis can be described, a person must know what proteins are made out of. There are four basic levels of protein organization. The first is primary structure, followed by secondary structure, then tertiary structure, and the last level is quaternary structure. Once someone understands the makeup of a protein, they can then begin to learn how elements can combine and go from genes to protein. There are two main processes that occur during protein synthesis, or peptide formation. One is transcription and the other is translation. Although these biological processes slightly differ for eukaryotes and prokaryotes, they are the basic mechanisms for which proteins are formed in all living organisms.