AIM

To test the pernicious anemia using the western blot assay and the SDS.

What is PAGE-SDS?

This is the method by which one gets to analyze the proteins in the electrophoresis method; in this case, we will use the technique to study the proteins in the human intestine to check for the insufficient B12 vitamin.

Introduction

it is the type of anemia caused by lack of vitamin B12 and also takes place where the body has insufficient red blood cells and, as known, the red blood cells provide the oxygen in the tissues through the organic hemoglobin. Pernicious anemia occurs where the intestines cannot absorb vitamin B12. Hemoglobin is a compound that combines with the oxygen and transports it to the tissues of the body. Hemoglobin levels of below 13.5 per 100 ml are anemia in males, while in females is 12.0/100ml of blood; there may be a slight variance from the sources. This depends on the laboratory data and reference. The term pernicious means injurious, destructive, or deadly. This one occurs when there is a slight disruption of the red blood cells in the hemoglobin levels. Lack of cobalamin in the digestive tract is the primary cause, which is mostly obtained from animal products. And it can be stored in the body as long as it can be, which means that the diet can be inefficient for long before the deficiency is declared. This type of anemia is commonly diagnosed in males of 60 and above though the small amount can be seen in the newborn.

Symptoms of Pernicious Anaemia

The symptoms have a relationship with any other oxygen deficiency condition and the deficiency of vitamin B12, which interferes with the nervous system’s proper functioning, which may bring the symptoms related to that of the nerve to break down. This may lead to the clumsiness, impaired judgment and coordination of the nerve transports, and lost energy. The patient may feel dizzy, which results from low oxygen concentration in the brain and the other parts of the body, leading to incomplete metabolism. Pale skin color can be seen as well; this is a result of bloodless rich in oxygen. The funny part there are some of the pernicious anemia patients diagnosed while patients will not be showing the symptoms (Yao, 2019). This type of anemia is commonly seen in the Caucasian people in north Europe more than the other racial groups.

DIAGNOSIS

On diagnosis, the first thing to be done is to collect the patient’s history and if there might be any story of infection linkage. Then from there are several laboratory tests done; the tests may include CBC, which is a complete blood count (Seage, 2019). When the lab result is out, the doctor may prescribe the drug or treatment.

TREATMENT

Pernicious anemia has its specialists, either the family doctor or the blood specialist, which is the Haematologist. The bone marrow biopsy is done in some cases in which there may be intensive destruction of the blood cells.

Materials

Polyachlyramide gel

Gel running buffers

Electrophoresis power supply

Molecular weight maker

Set of auto pipettes and tips

Microfuge tubes which are sterile

Disposable sponge

Ballpoint pen or pencil

Gel roller tool scissors

50ml falcon tube

METHOD

The polycliramide gel was pre-casted in the electrophoresis tank, and both reservoirs were filled with the tris-glycerine buffer

1. The stomach protein sample was then prepared by addition of 12 microliters of SDS buffer to a tube containing 48 microlitres of mouse stomach proteins
2. The mixing was thoroughly done by pipetting until the mixture was homogeneous, and the bubbles should be avoided at this stage

• The tubes were then sealed and left to stay at room temperature

1. The 5micrpliter was then loaded into a blotting machine
2. The apparatus was then connected to the power supply, and the gel was run at 200V for 40 minutes to 1 hour
3. The power supply was then turned off, and the gel removed noting the position of the protein and maker on the gel

• The help of the illustrator then opened cassette
• The gel was therefore removed and the gel placed in a plate containing the buffer just enough to immerse the gels

1. The cellulose was then transferred to a nitrocellulose membrane and a set up done on the base of the blot
2. When the transfer was done, the power supply was then put to the nitrocellulose membrane in the container
3. The results were then visualized

Experimental Results

Fig 1. Diagrams of a histological section of mouse stomach

Picture diagrams for positive and negative sections of mouse stomach

The negative control of immune-peroxidase staining gives visualization due to epitope and paratope reactions from similar components. Both the patients show negative on some of the tumor types.  Western blotting is observed, as shown in the above pictures for positive and negative control.

            Figure 2. diagram of colonic crypt

Both scanned hand-drawn low power diagrams of colon X40 magnification arrows show parietal and chief cells’ stomach glands within mucous cells.

Fig 3(a) hand-drawn diagram of the colonic crypt at high power magnification of X400. The lines show the gobbled cells and nucleus, together with primary gastric cells.

Fig. 3(b) the photo of high power colonic crypt under X400 magnification

Discussion

The polyacrylamide gel is used to analyze the DNA samples of the. In this process, there are several discussions we need to know using an individual chemical component of the research material and analyze. The PAG is used for embedding the medium and tissues the gel is composed of the following elements include the following: Chemical buffer; this buffer is used to stabilize the PH of the components of the gal within the gels itself, and in the buffer used in the electrophoresis when the buffer was we focused on the counterions and the gel resolution the different cushions are to be used as the anode or cathode buffers depending on the charges present.

The acrylamide gel is dissolved in water, so as there is slow polymerization of the acrylamide as it takes place slowly, which helps to join the molecules to the tail. The sodium dodecyl sulphate SDS is usually applied only when there is a need to denature proteins this is used as a method to denature the proteins to individual polypeptides the SDS is used to facilitate the constructive denaturalization, and this is the reason why our proteins are seen in the linear form as pour the report. The nucleic acid strain, which is the ethidium bromide, is the one used to allow the visualization of the DNA or RNA on the gel. The fluorescence nature of the EtBr under the UV light gives this nature. The ethidium bromide takes part in the process of intercalation. The thing one needs to understand about the ethidium bromide is that it is carcinogenic, and therefore care should be taken when using it in staining.

Protein Being Detected by The Sera from Pernicious Anaemia

The protein cited was confirmed to be having the immunoblotting reaction with a 70KD protein in the canine of the rodent. In the gastric cells of the mucosal in the intestinal walls, the mucosal cells are enriched with the 80to 90% of the parietal cells. There were the positive sera that were demonstrated by the micro-body evidenced in the immunofluorescence (Gilbert-Estelles, 2012). The positive sera micro-body from the parental cells with the unconfirmed parietal anemia was seen reacting the same with the 65-70KD proteins. Therefore, it confirms that there is a probability that there is the presence of the parietal cell, which occurs in the reaction after the gel’s loading.

Sub Unit of Proton Pump Corresponding to Reaction

The proton pumps that correspond with the proton pumps’ inhibition, which include the hydrogen ion pumps. It is as a result of the hydrochloric acid found in the gut. An example of such a reaction is in the ulcers where the omeprazole is used as a powerful proton pump. This is the reaction where the base i.e., omeprazole, blocks the h+/k+ ATPase and the proton pump activity. And the inactivation of the proton pump at a neutral PH. The v type ATPase proton pump is also seen taking place in the reaction, and it is a macromolecular complex made of 14 subunits grouped into two main functional domains.

Other methods to quantitate the antiproton pump

The other method that can be used to quantitate the level of the proton pump includes the immunoassay. In this aspect, the monoclonal antibodies mainly depend on the ATP, responsible for the secretion of acid in the gut. This immunoassay entails the absorption of the samples. Then there is the mediation of the gastric acid by proton, which may be translocating potassium ion-dependent adenosine triphosphate.

Preparation of biopsy specimens: The gastric samples for this type of biopsy are gotten from the Olympus. The biopsy specimens were then stored suspended in 1ml PMSF and then disrupted at the 2000rpm and then ten strokes. Then the homogenates sonicated in aliquots and supernatants then centrifuged for 30 mins at a speed of 20,000. The supernatants after the centrifuge were then applied with no more processing in the microplate wells. Finally, the measurement of proteins was then conducted by the SDS Lowry procedure.

Antigen-specific for in-HRP Conjugate

The HRP is mostly used to stabilize the HRP as the label proteins and the antibodies. The stabilization of superior antibodies is, therefore, seen in both low and high proteins.

Enzyme Conjugated to HRP Antibody.

The HRP conjugated antibody is there to amplify the signal when utilizing the catalyst. The alkaline phosphatase. The hrp phosphates are widely known in performing immunoassays like the Elisa tests and are efficient when the volumes are limited.

Sources of Errors

Since this is a lab and at this time we will be dealing with the chemicals and other microorganisms the error might arise in the following areas: Contamination of the buffers, low vision and not being able to interpret the color, error in measuring the volumes needed, and not following the manual keenly

Remedies

To get the accurate results, the following should be done: ensure the working desk is sterilized by ethanol 70%, ensure u get assistance to view the color to get clear vision, being keen when measuring volumes to avoid spillage when transferring, adhering to lab manual keenly, and every step recorded.

Conclusion

From the experiment, it is clear that the gel run shows some proteins responsible for causing the anemia. Maximum control of the enumerated conditions, it is advisable to take animal products, and also, one can take them in a multivitamin way in the form of tablets and syrup. The fluorescence pink shows the infections as per the images. The SDS method used here is widely used in the biochemistry field to extract and separate the proteins in the analysis. It was combined with the western blot assay to get to understand and check if there was a presence of a specific protein in the mixture. The method is also used in the medical diagnosis in testing for HIV patients, or in the proteinuria, and the SDS PAGE separates the proteins in case of HIV test. Western blot can also be used to check on patients’ antibodies present in the blood or the serum.

Works Cited

Gilbert-Estelles, J. B.-B. (2012). Roles of microRNAs in Gynecological Pathology. Current Medicinal Chemistry, 19(15), 2406-2413.

Parween, S. &. (2013). Image-Based ELISA on an Activated Polypropelene Microtest Plate-A Spectrophotometry-free Low-Cost Essay Technique. Biosensors and Bioelectronics, 287-292.

Seage, C. H. (2019). Receiving a Diagnosis of Pernicious Anaemia: Exploring Experiences of Relationships with Health Professionals. Journal of patient Experience, 2374373519883497.

Yao, P. (2019). Atypical Presentation of Pernicious Anaemia. Proceedings of UCLA Health 23.