Fibrinolytic Enzyme Lab Report

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

2. UV treatment increased the mechanical strength of the fibrin microthreads, decreased the swelling ratio, and decreased cell proliferation. UV treatment increased the mechanical strength of the threads the most at around 17 J/cm^2, and the UTS of the threads increased after treatment. Swelling ratio of the threads decreased from around four times more than dry threads to around 2.5 times more after UV treatment. Cell proliferation decreased because while the number of cells on non UV treated threads continued to

What impact does the illness have on the blood’s ability to clot? Include the role of vitamin K and the specific clotting proteins affected. Also discuss platelet production. Describe the test that is typically run to test the blood’s clotting ability.

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

- Coagulation inhibitors and Fibrinolysis : to Localize thrombus on injury and Prevent new thrombus formation.

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

In an article published in JACC: Cardiovascular Interventions, Doctors Madan, Halvorsen, Di Mario, Tan, Westerhout, Cantor, Le May, and Borgia explored whether patients experienced greater risk of undergoing angiography after the administration of fibrinolytic therapy. They concluded that there was not a serious risk of bleeding or death if they receive angiography within four hours of undergoing fibrinolytic therapy (Madan et al., 2015). They also suggest that the patient be moved a center that can perform PCI within 2 hours after fibrinolysis. This article suggests that although fibrinolysis can be success a patient should receive PCI treatment.

The kinetic profile of tube 1 is represented by a horizontal line in graph 1. Tube 2 contained 0.01ml of enzyme and graph 2 represents the kinetic profile of tube 2. It shows that the absorbance of tube 2 increased over time at an exponential rate. The amount of enzyme added to tube 3 was 0.1ml. The absorbance of this tube increased at a steady rate over time. Graph 3 represents tube 3 and its enzyme kinetic profile is indicated by the positive linear line. About 0.5ml of enzyme was added to tube 4 and graph 4 represents its kinetic profile. The absorbance increased over time, but at the end it began to plateau. Comparing the kinetic profiles of 4 tubes, the appropriate enzyme concentration needed for the assay was determined to be

The purpose of this lab was to test the biological activity of ConA by performing a hemagglutination assay. If ConA is active then agglutination will occur due to ConA’s free receptors being able to bind to the glucose residues on the sheep’s red blood cells. If ConA is not active then no agglutination will occur. To test the hemagglutination reaction, two types of ConA solutions were compared, a purchased control ConA solution in buffer as the positive control, and a purified solution of ConA in buffer previously purified in lab. Each solution was at a 2mg/ml concentration of ConA in ConA buffer, which is

Tissue plasminogen activator is a fibrinolytic drug which is used to treat thromboembolic disorders, such as ischaemic strokes. These agents initiate secondary fibrinolysis to occur; altering the haemostatic capability. The primary purpose of this agent is to clear occluded blood vessels within the systemic circulation (Bryant & Knights 2011, pp.534-536).

After the dogs had been anesthetized and given the injection of the endotoxin, blood was drawn. The blood was collected by use of a catheter in the femoral artery. However, for each sample, a new catheter was used. Between each collection of the samples, the artery had to be clamped using a Pott’s Clamp. The blood collected was then stored in a silicon tube. Nine units of whole blood were anticoagulated with one volume of 3.2% sodium citrate. The plasma was then separated by centrifugation for 30 minutes and stored in silicon test tubes at negative twenty

The precursor to thrombin is prothrombin, an inactivated protein produced in the liver. Upon injury prothrombin converts to thrombin with the purpose of catalyzing the conversion of fibrinogen to fibrin. The process changes the blood plasma soluble fibrinogen to fibrin, which is not plasma soluble. In order to do this the interaction removes two peptide chains from the ends of fibrinogen. Once this reaction is complete fibrin will begin to form a blood clot around the site of injury. While the interaction between fibrinogen is important it is not the only major

Introduction The purpose of this lab was to find the concentration of protein in each solution by establishing a standard curve and to determine the protein concentration in a BSA sample that had an unknown concentration. A spectrophotometer was used to measure the absorbance in solutions containing protein. The spectrophotometer stipulated the absolute protein concentration in the solutions. Hydrated copper sulfate, potassium hydroxide, and potassium sodium tartrate are the crucial components in the Biuret reagent (Biuret Test, n.d.).

Figure 1. Enzymatic activity was measured by adding 200 microliter’s of 100mM citrate buffer at different temperatures to 10 separate tubes with two tubes for each temperature ranging from 4 to 70 degree’s Celsius and 250 microliter’s of 5mM p-nitro phenol phosphate. The solutions were then transferred to a water bath for 5 minutes, and the acid phosphatase enzyme was added at certain intervals (30 seconds) after each addition. Once the solutions were made the group of solutions were analysed in the spectrophotometer twice for each temperature and using Beers law the enzymatic activity was calculated, mean values of enzymatic activity were then calculated. The graph was then created as shown above, mean enzymatic activity vs temperature in

Six plastic cups were obtained and labeled 0.2 M, 0.4 M, 0.6 M, 0.8 M, and 1.0 M. Next, each of the six dialysis tubes were knotted on one end and filled with the sucrose samples, and then tied off. These samples were then dried by patting with a paper towel, weighed and placed into their corresponding cups. The mass of each sample was recorded in table 2 on the data sheet. Each cup was then