Molecular Basis of Pernicious Anaemia Lab Report

Molecular Basis of Pernicious Anaemia - Lab Report Example This process is important because it is a requirement needed for the maturation of red blood cells, putting forward a relation with the disease2. Parietal cells are found in the gastric glands where there major function is to produce hydrochloric acid that is involved in the first stages of digestion. For gastric acid secretion, the gastric H positive or the proton pump enzyme that consists of ninety five kDa alpha subunit and a sixty to ninety kDa alpha subunit3. Auto anti bodies contained in serum from different patients having pernicious anaemia react with either or both of the alpha and beta subunits of the gastric proton pumps. Knowledge about this antibody response provides a useful diagnostic tool for the pernicious disease. Also, the most widely used method for analyzing protein mixtures is by gel electrophoresis. The most commonly used type of gel is the SDS polyacrylamide. It essential to realize that proteins are solubilized in sodium dodecyl-sulphate (SDS) , an anionic de tergent that binds to the hydrophobic regions of proteins causing them to unfold hence acquiring an overall negative charge. Inclusive is a reducing agent that is included to disrupt any disulfide bonds that are within the bonds. Avery good example of a commonly used reducing agent is the 2-Beta mercaptoethanol. On electrophoresing the proteins through a polyacrylamide gel, the negatively charged proteins migrate towards the anode and separation done with regard to their size, with smaller proteins moving the furthest. It is possible to perform an analysis of the size of proteins in the mixture if molecular weights of running proteins are known. This report tests antibody from the serum samples of different patients and the results to be used to identify if any of them reacts with gastric proton pump. The results acquired from the tests will hence provide a guide to finding a diagnosis of pernicious anaemia. For accurate results it is important to use two different methods which wil l determine the presence of anti-proton pump antibodies in serum samples4. Method one (Practical three) Separate proteins extracted from mouse stomachs by SDS poly acrylamide gel electrophoresis, and transfer the proteins to a nitrocellulose membrane. Stain a section of a mouse stomach with haemotoxylin and eosin so that it becomes familiar with the structure and cells types within the stomach. It is essential to realize that the cells in the mouse stomach are similar to those of a human’s anti-proton pump antibodies. For this reason, they cross react with the equivalent mouse proteins. Method two (practical 4) Perform Western Blotting on the gastric proteins using sera from patients who may or may not have pernicious anaemia. Western blotting has this procedure. First, an individual acquires the SDS-PAGE of mouse stomach proteins. These proteins are transferred to nitrocellulose membrane by electrophoresis. The membrane is stained with Ponceau S so as to confirm protein tran sfer from gel to membrane. After that process, the membrane is de-stained and then stored in blocking buffer until next usage. Use H19 to incubate strips of membrane and then wash the membrane strips and incubate in blocking buffer. The membranes are incubated in secondary anti-bodies and in luminous detection buffer. Lastly, the markers are reassembled and test strips are visualized with Chemi -doc system5. Also, perform immune peroxidase staining of mouse stomach