Thursday, April 4, 2019
Protein Determination Lab Report
Protein De confinesination Lab give notice (of)IntroductionWithin protein biochemis purify, analytical procedures and experiments have been designed to de full termine accurate measures of the protein dousing in a precedent. A variety of protein assays be organism and have been developed to be used in laboratories to analyze a solution and to quantify protein from a biological specimen. For certain dumbnesss, protein assays such as the Bradford and the Lowry method willing have advantages and disadvantages, for instance a uplifteder cost but the results have a greater trueness and are produced expeditiously (Becker, Caldwell and Zachgo, 2011). Protein assays are essential to determine if proteinuria is a result of glomerular or tubular manifestation, indicating serious health crookednesss. The protein white is the most abundant in piddle compared to that of other proteins, and so is the standard most common protein measured (Vogel, 2008).For decades, protein quantificatio n has been an important laboratory technique that extracts, purifies, characterises and analyses protein concentrations throughout human biology, from enzymatic activity to clinical testing of urine samples. In many parts of the world, such as Australasia in New Zealand, routine checks are available to measure egg white levels in the urine to aid in the diagnosis of renal disease (Martin, 2011). Diagnosing disease through protein quantification is a long-lasting technique, ancient experts would try to identify egg white in the urine through a technique known as the foam-test, which is too still carried out today to detect glomerular disease. For this reason, protein quantification is a crucial diagnostic tool in early detection of disease, classification of disease and assessment of prognosis (Pisitkun, Johnstone and Knepper, 2006).Blood plasm is comprised of a crew of proteins to aid in various factors including the maintenance of kind plasma volume and/or a protein transport er. Of the stemma plasma, albumen makes up 60% of plasma protein, and is the most copious protein to be measured in the plasma (Higgins, 2005). In healthy patients, the albumin concentration in the urine should be exceptionally low, between 20ug/ml and 30ug/ml are considered a normal level. However, any reading above 30ug/ml could be an singularity that the person could have microalbuminuria or macro-albuminuria which increases the risk of a be sick of health conditions (Hasanato, 2016).To determine irregular concentrations of albumin in the urine requires an accurate screening process, this is an essential diagnostic tool for the early detection of forward-looking kidney disease and a cardiovascular risk marker. The initial assessment uses proteinuria uranalysis on patients exhibit symptoms of chronic kidney disease (CKD). For instance, a clear characteristic that a patient with diabetes mellitus could develop Diabetic nephropathy is through an increase in the albumin in th e urine. Non-diabetic patients that develop a protein concentration in the micro-albuminuria surface area of above 30ug/ml are still at risk of CKD progression and higher CVD mortality risk. (Carter et al., 2006) albuminuria can affect people with or without diabetes, male or female but can more and more worsen with age. (Fraser et al., 2017) Several sources, including the Heart Outcomes Prevention Evaluation study, found a correlation between abnormal albumin levels detected in a dipstick test and Cardiovascular diseases such as hypertension and strokes. A recent study found that from 48,000 participants, the presence of microalbuminuria was shown to increase stroke risk by up to 90% than that of normoalbuminuric individuals (Delles and Currie, 2013).For the protein determination laboratory experiment, the aim of the albumin assay was to determine the absorbance care for of the 5 patient urine samples with the high and normal stops. To achieve the aim, three objectives were imp lemented the first step was to create an accurate turn off of albumin which aided in the second and third objective to determine the quantity of proteinuria in each sample, followed by a diagnosis of the patients from the urine samples.MethodologyUsing the Bradford Assay procedure, 8 standards are generated with the protein concentration from 0ul in standard 0, 25ul, 50ul, 75ul, 100ul, 250ul, 500ul and 1000ul in standard 7, Adding water to labelled microtubules with Bovine Serum egg white (BSA) 1mg/ml (1ug/ul). Duplicates of 100ul of each solution are then added to test tubes with 5ml of Bradford regent and incubated at room temperature for 10 proceedings before being recorded at 595nm in the spectrometer. These set are recorded into excel, a calibration graph is formed through calculating the average, producing the R2 assess. The calibration graph calculates the M value in y=mx this is used to determine the protein concentrations of each sample. After the duplicated samples h ave been left for 10 minutes containing 100ul of each patients sample and 5ml of Bradford regent, these samples are read at 595nm in the spectrometer giving the Y absorbance values through light absorbance. In excel, 6 anomalies were removed from each data set before being analysed, averaged and the standard deviation being calculated. Using the average and x=m/y equation, a agglomerative column bar chart was formed showing the quantification of albumin protein concentration showing the +/- standard deviation.ResultsThe protein albumin in the urine is an essential marker for the diagnosis and prognosis of certain diseases. water tests, which contain high levels of albumin are usually the first steps in determining illness and disease. Certain symptoms that a patient shows can be a clear distinction that albumin levels are high. Testing of these albumin levels can then significantly help determine illness. It is paramount for this reason to quantify albumin protein concentrations in samples to aid in the keep onion of morbidity and mortality rates worldwide. In this study, five patient samples were well-tried for albumin against a high controlled and normal controlled sample to show the importance of albumin in urine. Upon the results, shown in figure 1 and 2 a diagnosis could be made for the individuals.To critically analyse the calibration graph in figure 1, the R2 trend line label must have a value of 0.95 to orient a good fit and a linear line for the data, the R2 label has a value of 0.99031 which follows this trend. It can be seen from the calibration graph has a linear relationship as the concentration of albumin doubles, the absorbance 595nm also doubles.In figure 2, the normal controlled and high controlled sample give a clear indication of how healthy the albumin concentrations are that are found in each patient sample. Although the high controlled sample has an albumin concentration of over 300ug/ml representing macro-albuminuria, patient 1 an d patient 4 have like abnormal protein levels in the serum showing microalbuminuria. Furthermore, patient 2 and patient 5 are also classed as having microalbuminuria as the concentrations are over 30ug/ml, however the levels are not extreme, and are not an indication of chronic kidney disease. long-suffering 3 has a similar concentration to the normal controlled sample the patients kidneys have close no protein and are classed as healthy.DiscussionIn a healthy urine sample, normal protein albumin concentrations are between 20ug/ml and 30ug/ml, this can be seen in Figure 2 with the Normal control having a value around 30 and for patient 3. However, protein levels from 30ug/ml to 300ug/ml are considered microalbuminuria patient 1, 2, 4 and 5. The High Control Protein has a value in the 340 regions, this is known as macro albuminuria (Hasanato, 2016). persevering 1 35-year-old female. ovalbumin protein concentration is approximately 210ug/ml, abnormal level of protein is extremely higher than the normal range and can indicate preeclampsia. Women who experience preeclampsia during their pregnancy are more likely to experience complication than those with normal levels of protein, headaches are a clear indicator that the lady is showing signs of preeclampsia (Hawkins et al., 2015). It is essential that her blood pressure is monitored to prevent hypertension and sent for various blood tests to observe her kidney and liver function (NHS 2). uncomplaining 2 50-year-old male. Albumin protein concentration is around 80ug/ml which is classed as microalbuminuria. Body mass index, waist circuit and high levels of protein in urine are linked with the formation of kidney stones (Garimella et al., 2015). The symptoms that patient 2 is experiencing suggest that he is suffering from a large kidney stone. Highly recommended that he is sent for blood and urine tests to check for transmission (NHS 1).Patient 3 40-year-old female. Albumin Protein concentration is roughly 25 ug/ml which indicates very little protein in her urine, concentration classed as normal as similar value to the Normal Control protein.Patient 4 23-year-old female. Albumin Protein concentration is in the region of 275ug/ml, classed as very high and could indicate albuminuria. Early diagnosis could prevent the patient from experiencing hypertension, type 2 diabetes and coronary heart disease (Yadav et al., 2016). The symptoms that the patient is experiencing could indicate a urinary Tract Infection, there is a strong correlation in many studies that demonstrations how a UTI can blow the glomerular involvement increasing the levels of albumin in the urine (Gonzlez Rodrguez et al., 2009).Patient 5 70-year-old. Albumin protein concentration boarding 60ug/ml which isnt significantly higher the normal range but still classed as micro-albuminuria. Patient showing signs of still recovering from influenza, immune system is weakened due to age and can beat longer to recover. Renal functi on decline can occur with age and within the elderly generation, which could indicate the abnormal levels of albumin in his urine (Funk et al., 2016). Should be monitored regularly, a review of his medication that may coif a decline in his renal function and put on antibiotics if symptoms of flu continue.ReferencesBecker, J., Caldwell, G. and Zachgo, E. (2011). Biotechnology. maiden ed. San Diego faculty member Press, p.119.Carter, J., Tomson, C., Stevens, P. and Lamb, E. (2006). Does urinary tract infection cause proteinuria or microalbuminuria? A systematic review. Nephrology Dialysis Transplantation, 21(11), pp.3031-3037. usable https//academic.oup.com/ndt/article-lookup/inside/10.1093/ndt/gfl373. conk accessed twelfth March 2017.Delles, C. and Currie, G. (2013). Proteinuria and its relation to cardiovascular disease. 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