1.4.3 Thermostability of Lysozyme on SDS-PAGE
To determine the thermostability of lysozyme enzymes, Akinalp et. al. (2007) exposed the supernatant of various recombinant bacteria to various temperatures (from 37C to 100C) for 15 min followed by centrifugation at 15,000 rpm to remove denatured proteins. The supernatant was then mixed with an equal volume of trichloroacetic acid and the protein collected by centrifugation. Protein analysis was then performed using a denaturing polyacrylamide gel (SDS-PAGE, 12% (w/v)). After electrophoresis, protein bands were visualized by a coomassie blue staining. As a result, the thermostability of the lysozymes from the recombinant bacteria was found to be different. The lysozyme expressed by S. salivarius subsp. thermophilus cells has high thermoresistance and was not denatured at 70C for 15 min. In contrast, the enzyme expressed by L. lactis and E. coli cells was easily denatured when exposed to the same temperature.
1.4.4 Immunoassays
The lack of sensitivity and long assay times of the turbidimetric and lysoplate techniques has led to the development of various immunoassay methods for the determination of lysozymes. Immunoassays rely on the reaction between the target analyte and a specific binding molecule of biological descent (the antibody). They can produce both quantitative and qualitative results and have shown considerable improvements in sensitivity (Ekins andand Chu, 1997). For example, Porstmann et. al. (1989) developed an enzyme immunoassay for the detection of lysozyme in patients with Crohn’s disease and rheumatoid arthritis. Urine samples were taken from patients and were tested using three variations of the same method. The method showing highest sensitivity involved pre-co...
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...ed with a horseradish peroxidase conjugated secondary antibody. The total test time is 4 ¼ hours (http://www.funakoshi.co.jp/data/datasheet/BTI/BT-630.pdf).
Similarly, a company Orgentec (Mainz, Germany) supplies a kit known as Anti-Lysozyme kit for lysozyme in serum and plasma. This test can process 96 patient samples at a time, only requires 10 μl of sample and involves a plate pre-coated with antibody. The antigen from the patients’ sample is added along with a horseradish peroxidase conjugate and then followed by TMB (a color substrate). The reaction is stopped by adding hydrochloric acid; the total time for this assay is two hours, due to the plates being purchased pre-coated (http://www.orgentec.com/products/pdfs/ELISA_en_IFU_ORG_526.pdf). All of these kits have the advantage of conducting analysis on several patient samples using minimal biological fluid.
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
The shape of the molecules is changing and so the enzyme molecules can no longer fit into the gaps in the substrate that they need to and therefore the enzymes have de – natured and can no longer function as they are supposed to and cannot do their job correctly. Changing the temperature: Five different temperatures could be investigated. Water baths were used to maintain a constant temperature. Water baths were set up at 40 degrees, 60 degrees and 80 degrees (Celsius). Room temperature investigations were also carried out (20 degrees).
Bioaffinity chromatography is a type of affinity chromatography in which biological compounds such as immunoglobulin-binding proteins, enzymes, lectins, carbohydrates, avidin/biotin system and antibodies are used as ligands (Hage, 2006). Immunoglobulin-binding proteins, namely protein A which is produced by Staphylococcus aureus and protein G which is produced by streptococci, are the ligands that are used in the vast majority of bioaffinity chromatographic applications (Tetala and van Beek, 2010). However, enzymes and enzyme inhibitors can also be used as affinity ligands (Hage, 2006). Immobilized enzymes are widely utilized in many applications, concerning pharmaceutical and food industries. Furthermore, they are used in order to purify enzyme inhibitors, as well as for the removal of impurities from unprocessed extracts. In a similar way, enzyme inhibitors can be utilized for the purification of enzymes from crude extracts (Tetala and van Beek, 2010). The immobilization of enzymes on monolithic stationary phases enables them to be used in a wide range of applications concerning bioaffinity chromatography (Petro, Svec and Fréchet, 1996).
Several tests may be performed on patients to determine the cause of lymphatic damage and elephantiasis. A definitive diagnosis of lymphatic filariasis is done through the identification of the microfilariae in blood. Samples of blood are taken at night. Other test used for diagnosis is immunodiagnostic test; it can identify the cause of the symptoms based on the detection of antigens of Wuchereria bancrofti. This test is highly specific and sensitive, blood samples do not have to be taken at night (Seppa
To conclude, the ELISA method is effective in detecting different diseases and is an ideal method for screening of diseases and toxins also. There are also a wide variety of ELISA methods that can be used depending up on the antibody and antigens that need to be detected. Also, as mentioned, they are highly sensitive which means they can give accurate and reliable results. They are also relatively inexpensive and do not require a high degree of skills or expensive equipment to use, but can be prone to experimental error if procedures are not followed.
Biology literally means "the study of life". Biology is such a broad field, covering the minute workings of chemical machines inside our cells, to broad scale concepts of ecosystems and global climate change. Biologists study intimate details of the human brain, the composition of our genes, and even the functioning of our reproductive system. Biologists recently all but completed the deciphering of the human genome, the sequence of deoxyribonucleic acid (DNA) bases that may determine much of our innate capabilities and predispositions to certain forms of behavior and illnesses. DNA sequences have played major roles in criminal cases (O.J. Simpson, as well as the reversal of death penalties for many wrongfully convicted individuals), as well as the impeachment of President Clinton (the stain at least did not lie). We are bombarded with headlines about possible health risks from favorite foods (Chinese, Mexican, hamburgers, etc.) as well as the potential benefits of eating other foods such as cooked tomatoes. Informercials tout the benefits of metabolism-adjusting drugs for weight loss. Many Americans are turning to herbal remedies to ease arthritis pain, improve memory, as well as improve our moods. Can a biology book give you the answers to these questions? No, but it will enable you learn how to sift through the biases of investigators, the press, and others in a quest to critically evaluate the question. To be honest, five years after you are through with this class it is doubtful you would remember all the details of meatbolism. However, you will know where to look and maybe a little about the process of science that will allow you to make an informed decision. Will you be a scientist? Yes, in a way. You may not be formally trained as a science major, but you can think critically, solve problems, and have some idea about what science can and cannoit do. I hope you will be able to tell the shoe from the shinola.
Morse, Stephen A., et al. "Detecting biothreat agents: the laboratory response network." ASM News-American Society for Microbiology 69.9 (2003): 433-437.
In this test the person is exposed to a suspected allergen under controlled circumstances. It may be included in the diet or by breathing in the allergen. This should be done only by a doctor as it may provoke severe allergic reactions.
16. Describe two evolutionary consequences if the process of crossing over in meiosis ceased to occur. If crossing over in meiosis ceased to occur there would be less genetic variations and no diversity among a species. This would essentially mean that a species would not be able to adapt to an issue that could arise in the future, meaning that its species could potentially become extinct due to climate change or other arising events.
Schizophrenia is a complicated, mostly permanent psychological disorder involving a disturbances in the relation amongst thought, emotions, and behaviour, leading to defective perception, inappropriate actions and feelings, withdrawal from reality. The National Mental Health Commission makes 10 recommendations, including reducing the use of restraint, seclusion and involuntary treatments. Recommendation 6 states, “There must be the same national commitment to safety and quality of care for mental health services as there is for general health services.”
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
Analysis of a total of eighteen samples (six samples at each of the three test levels) which were prepared by adding known amounts of standardized chlorine solution to 0.1% sulfamic acid collecting solution.
The enzyme used in this lab is catalase. Catalase has a molecular weight of approximately 240,000 daltons and contains four polypeptide chains, each composed of more than 500 amino acids. This enzyme occurs universally in aerobic organisms. One function of catalase within cells is to prevent the accu...
Disadvantages of using antibody are that the binding capacity is strongly dependent on assay conditions such as pH level and temperature and this assay’s interaction between the antigen...