Name: Course: Tutor: Date: PERNICIOUS ANEMIA Introduction Pernicious anemia is a disease caused by the loss of the parietal cells of the gastric mucosa. These cells produce an essential protein known as intrinsic factor. The intrinsic factor helps in vitamin B12 absorption…
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People lacking the absorption ability of vitamin B12 remain with the disease for the rest of their lives. Parietal cells found in the gastric cells are responsible for production of hydrochloric acid which is important in digestion as it provides a favorable medium for enzyme reaction. The serum found from patients having this disease react with ? and ? sub units. The antibodies are used for diagnostic purposes. This experiment involves testing of serum of antibodies from various patients to determine if they react with the sodium pump which contains ? and ? sub units. This will help in diagnosing pernicious anemia. A mouse is used in this experiment since its stomach structure is similar to that of a man. A primary antibody and a secondary antibody are also used in this experiment as they react with the mouse antigens. Anti proton pump of a human being can also cross react with proteins of the mouse making the mouse a favorable specimen for the experiment. From the experiment we expect antibody response to help us diagnose pernicious anemia. Aims The aim of this experiment is to determine whether samples from patients contain antibodies and also to diagnose patients having the disease through Western blotting; Immuno histochemistry using the gastric proton pump. Materials and Methods The materials used includes; SDS polyacrylamide gel (80%, resolving gel 40% upper stacking gel), Gel running buffer (25 mM Tris, pH 5.3, 0.192M Glycerin, 0.1% SDS), Gel apparatus, power supply, mouse stomach protein,5? SDS sample buffer (0.315 Tris, pH 6.8, 25% glycerol 10% SDS, 5% 2-beta mercaptoethanol, 0.025% Bromophenol blue), Protein MW standards, heat block, Gel loading tips, transfer apparatus, transfer buffer (25mM Tris, 192mM glycine,20% methanol), nitrocellulose membrane, filter paper, 0.1% Ponceau in1% acetic acid, 0.1M NaoH, TBS Tris buffered saline, Blocking solution TBS containing 5% skim milk powder. Materials required to obtain a mouse stomach include; a slide containing section of mouse stomach, xylene, hemoglobin, acid alcohol, Scott’s tap water, eosin, DPX mounting media and cover slips. Procedure to obtain mouse stomach morphology The slide containing mouse stomach is incubated in xylene and ethanol for two minutes respectively. After this the slide is rinsed in tap water for 30 seconds. The slide is then incubated in the hemoglobin for 2 minutes. The slide is rinsed again in tap water for 30 seconds after which it is placed in 1% acid alcohol for 3 seconds after which it is rinsed again in tap water for 30 seconds. The slide is incubated in Scott’s tap water for 30 seconds after which it is swashed in water for 30 seconds. The slide is then placed in eosin for 4minutes and excess is blotted off by a paper towel. The slide is then placed in 80% ethanol and again 90% after which the slide is incubated in ethanol for 2 minutes then allowed to air dry. A drop of DPX mounting medium is placed on the section and then covered by a cover slip. An observation is made under the microscope and diagrams made as follows. Fig 1 showing a mouse stomach with Haematoxylin To prepare the stomach protein sample, 200µl of protein is placed into a microfuge tube and 50µl 5? SDS sample buffer is added. The marker and the protein sample tubes are spine for a few seconds to bring the liquid to the bottom of the tube. The MW markers and proteins are then loaded using a special gel loading tips. The apparatus is then connected to a power supply of 200V and run for 1 hour until
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