Pernicious Anemia and Techniques Used in Molecular and Cell Analysis - Lab Report Example

PERNICIOUS ANEMIA Submitted by: Student name + registration Subject School Tutor Introduction Anemia is the state where a person has limited quantity of erythrocytes in his bloodi. An important aspect of erythrocytes is hemoglobin which is used to transport oxygen in the body. There are various forms of anemia among them Pernicious Anemia. This is a disease caused by failure of the digestive system to take in vitamin B12. Consequently, megaloblasts which are ingrown erythrocytes flow in blood although they cannot act like erythrocytesii. Western blotting or protein immunoblot is a technique used in molecular and cell analysis. It is used to analyze proteins supported by their capability to combine to precise antibodies. The uniqueness of antibody- antigen relations facilitates recognition of intended protein in middle of composite protein assortment. The commonest way to do this test is through iBlot probing. In this technique, iBlot Western detection and transfer kit is used. The kit is preferred as it is reasonably priced and easy to use. It is also available in the market. Membranes are cleansed with distilled water to be used in the experiment. IBlot is assembled and air bubbles removed while membranes are placed inside it. The machine is started and stops at the completion of each stage. It is then restarted for two other consecutive times with fresh membranes. Each time, the process is repeated. The results are then noted down for analysis after the final testing. Immuno-histochemistry is the technique used to identify existence of particular antigen in organ sample or antibodies in a serum sectioniii. After the free serum is washed, the sample is nurtured with a derived antibody unique for person Ig molecules. This antibody is modified as enzyme that transforms a substrate into a dyed result in parts of the organ sample where basic antibodies are confined. The results of the experiment are recorded down for analysis. Aim The main objective of this experiment is to test the presence of pernicious anemia in human beings. This will help in diagnosing the patient if the disease is found. It will act as a basis for carrying out further test and consequential treatment of the patient. Method Experiment 1: iBlot Probing experiment Step 1 We removed the membrane strips from TBS and rinsed them with distilled water. Later, we soaked the matrix on a crystal clear paper using 5 ml of 5% skim milk jamming solution. We then assembled detection stack in an iBlot machine and gently sloped out bubbles of air in each layer. A sponge was placed in the iBlot cover with the metal handle in the upper right spot. We then closed the lid and the clump shut. We selected program 9 on the iBlot and pressed start. We waited for two minutes. During this period, we soaked a second fresh matrix using 2.5 ml of the patient’s sample on a see-through sheet. The machine beeped. We opened its cover and removed the top stack. We then stored it back in the red plastic tray provided. We then removed the matrix and inserted spacers in between the membrane strips. We separated the membranes according to the patient’s sample. Later, we covered these membranes with personal matrix by means of pre-soaked sick person’s control matrix. Step 2 We rolled out the air bubbles in each layer of matrix. We took great care to ensure that the matrix from one group did not cross to the other group and contaminate the specimen. We replaced the top stack and rolled out the air bubbles. Later, we closed the iBlot lid and clump shut. We then pressed start in the machine. A the primary incubation continued, we soaked the final soak a final fresh matrix in a 5 ml derived antibody onto a crystal clear paper. The machine beeped the second time after the step was completed. We opened the lid and removed the top stack. We stored it back as usual, in the red tray provided. Step 3 We removed the used matrix and also the spacers. We replaced them with pre-soaked derived antibody matrix. We then rolled out the air bubbles. We replaced the top stack and further rolled out air bubbles. We clump shut the machine and pressed start. The machine beeped at the completion of the program. We opened the lid and removed the sponge, matrix and top stack. Later, we discarded them. With the aid of forceps, we removed the membrane. Then we discarded the bottom stack and turned off the machine. Washing the Membranes We then proceeded to wash the membrane strips inside TBS three times for a period of 5 minutes using the trays provided. Then, we prepared the Western detection Prepare Western detection mixture by combining solutions A and B in a 1:1 ratio i.e. 0.001 ml per each strip. The membrane was nurtured in a concoction of Chemiluminescent recognition solution for a period of 5 minutes. The membrane was afterwards placed in a plastic pocket to be imaged using a CCD camera. We placed the plastic bag containing the membrane into Chemidoc apparatus for detection and analysis using the light emitted from the substrate. The image was placed on CloudDeakin. We then cropped two images for use during analysis. After the completion of experiment 1, we proceeded to carry out experiment 2. Experiment 2: Immuno-peroxidase Staining The slide was incubated in xylene for 2 minutes. Then it was incubated in ethanol for another 2 minutes. It was later rinsed in tap water for 30 seconds. The slide was then incubated in 0.3% of hydrogen peroxide for 30 minutes to remove the autogenously peroxide action. It was then washed in PBS for 2 minutes. Later, it was incubated for 30 in 1% of BSA inside PBS. This was to prevent min to block non-specific combination of proteins. We removed the blocking solution. Later, we added 0.001 ml of the sick person’s Remove the blocking solution and add 100 µL of the patient’s blood specimen to the segment and nurtured it for 20 minutes. Then, we washed the slides three times for two minutes each inside PBS. Then we added to the segment 0.0005 ml of sheep anti-human Ig HRPO mixture which was diluted at a ratio of 1:300. We then incubated it for forty five minutes. We then washed the slides three times for two minutes each inside PBS and later briefly rinsed them in water. Subsequently, we added 0.001 ml of DAB substrate into the section and nurtured it for ten minutes. We rinsed the slides in PBS and dipped them into haematoxylin for a period of three minutes. We then washed these slides for thirty seconds in water. We later nurtured the slides for two minutes in ethanol. We placed a small drop of DPX mounting medium into the segment. Then we covered it with a cover slip. We then cleansed the excess mounting standard from the slide. We finally examined the slide under a microscope to resolve if parietal cells were stained. We prepared the diagram of stomach segment. We also got the results of other groups for further analysis. Results The following images show the parts of the rat: Image 1: Internal section of a mouse Image 2: Parts of the rat stomach. Our results for the experiment were as shown below. On Thursday evening, we obtained the following: Figure 1: Results after the first step of experiment. This was at the beginning of the experiment and the blots had not travelled. The pre-transfer gel was stuck at the base. This problem could be as a result of shift stack assembly being left for more than thirty minutes in the machine after transfer was complete. We should have disassembled the machine as soon as thirty minutes were over to avoid this problem. The following morning i.e. Friday morning, we obtained the following results from our experiment: Figure 2: Results after second step of experiment. The results were faulty as the air bubbles were still present in the membrane and the gel hindering transfer of the proteins. This led to empty sports on the membranes. We should have completely rolled out air bubbles to prevent this problem. In addition, the few protein bands available were distorted. This is due to un-uniform electric spectrum that was created in the region of the wells. To prevent this occurrence, we should have flattened well protuberances on the gel appropriately using the staining roller. In the evening we obtained the following image from our results: Figure 3: Results after the third step of experiment. The results were still faulty as the air bubbles were also present in the membranes and the gel thus hindering transfer of the proteins in the membranes. This caused empty sports to be formed on the membranes. We should have completely rolled out air bubbles to thwart this problem in the experiment. The few protein bands available were distorted due to un-uniform electric spectrum that was created in the region of the wells. This occurrence could have been alleviated by flattening well distensions on the gel properly with the help of the staining roller. On Monday morning, we had the following results from our bench: Figure 4: Results after the fourth step of experiment. The outcome had empty sports on the membrane. Air bubbles could not be rolled out completely thus held back. Air bubbles should have been rolled out completely to prevent this negative occurrence in the experiment. The final results as obtained on Monday evening we as follows: Figure 5: Results after the final step of the experiment. Protein bands were distorted on the membranes due to inconsistent electric band which was formed in the area of the wells. The incidence should have been alleviated by evening out well the swellings on the gel as it should be with aid of the staining roller. Our results for the experiment were all faulty hence we had no option but to use the technical staff results for analysis. The staff results were as shown below. Figure 6: Technical staff results after final step of experiment Discussion For a patient suffering from pernicious anemia, the expected protein size is between 65 and 90 kDa. Therefore in this experiment, for a positive outcome of the test, we expected a protein size of 65-90 kDa. This corresponds to beta (β) subunit of the abdominal H/K-ATPase proton power. From the results, it is evident that the patient P1 and P2 were suffering from pernicious anemia. Their protein sizes corresponded to the disease’s kDa (60-90 kDa) (figure 6). Therefore the experiment had positive results. The derived antibody for the experiment was raised in a sheep and it is specific for parietal cell antigen. It is conjugated using Horseradish Peroxidase enzyme denoted as HRP. Another method that can be used to test and give the quantity level of anti-proton pump is ELISAiv. ELISA stands for Enzyme Linked Immunosorbant Assay. It is done on a 96-well salver that allows excellent outcome. The lower par is encrusted by protein that binds antibody to be measured. The blood sample is allowed to clot. Later, centrifuge is done to get plain sera known as basic antibodies. It is the nurtured in well where every one of them has special serum. Affirmative and downbeat controls are integrated into the ninety six samples for testing. Later, the sera are taken away where washing of faintly supported antibodies is done through a sequence of shield rinse. For identifying combined antibodies, derived antibody is supplemented into every one of the wells. Derived antibodies should have bonded to each and every human antibody. It classically fashioned in rodents. Enzymes like basic phosphates or peroxidase are joined to derivative antibodies. They convert chromagens to painted results. When culturing is over, the resultant antibody suspension is eliminated where slackly joined antibodies are cleaned same as previously. The ultimate activity is adding enzyme substance with formation of tinted result in wells. After enzymatic response has completed, complete cover is situated in reader for the ocular concentration of each well to be established. Quantity of tint formed is directly comparative to sum of most important antibody joined to proteins at the basement of the wells.  References 1. David, C.P., Pernicious Anemia and Vitamin B-12 Deficiency, MedicineNet.com, 2013, retrieved 7 October 2014 from http://www.medicinenet.com/pernicious_anemia/article.htm 2. Gersten, T., Pernicious anemia, MedlinePlus, 2014, retrieved 7 October 2014 from http://www.nlm.nih.gov/medlineplus/ency/article/000569.htm 3. National Cancer Institute, Immuno-histochemistry, National Cancer Institute, 2014, retrieved 7 October 2014 from http://www.cancer.gov/dictionary?cdrid=653117 4. Life Technologies, Protein Handbook, Life Technologies, 2013, p. 34, Retrieved 7 October 2014 from http://www2.lubio.ch/uploads/tx_pdforder/Novex_ProteinHandbook-Final.pdf Read More