LIFS 2720 – Introductory Biochemical Laboratory Lab Report 4 Serum Electrophoresis Using Cellulose Acetate Name: CHAN Kin Yan ID:20094186 Group No. 9 Date of Experiment: 1st March 2012 Abstract Electrophoresis is a useful tool to separate components in a mixture based on their charges and differential mobility. Proteins are electrically charged. When put under an electric field, proteins with different mobility migrates towards the electrode at different speed. The rate of mobility is determined by the balance between the driving force and the frictional force.
The higher the rate of mobility, the closer the serum proteins move to the anode. In the experiment of cellulose acetate zonal electrophoresis, barbital buffer and bromophenol blue were used in the steps of sample loading and staining of membrane. The result showed that serum albumin has the highest concentration, followed by ? Globulin, ? Globulin and ? 2 Globulin indicated by the colour intensity of the bands and peaks on the chromatogram. Also, the smaller the protein, the nearer to the anode due to the smaller resistance. So, serum albumin had the smallest size and ?
Globulin had the largest size accordingly. 5 peaks should be observed in the chromatogram but our result had only 4 peaks. It was believed that the peak for ? 1 Globulin was missing as it had low concentration and similar size with ? 2 Globulin, so the peak was not visible. Various types of diseases like Multiple Myeloma and Sickle Cell Anemia can be diagnosis by many different forms of electrophoresis in laboratory. Introduction Separating serum proteins is a useful diagnostic tool and it is also a way to monitor clinical progress. Serum proteins are proteins that present in blood erum. They serve many functions, including transport of lipids, hormones and vitamins in the circulatory system, etc. Albumins, globulins, fibrinogen, regulatory proteins and clotting factors are the five families in serum protein. In this experiment, only albumins and globulins were focused. 55% and 38% of blood proteins contains serum albumin and globulins respectively. Serum albumin maintains the osmotic pressure of plasma so as to assist the transport of lipids and steroid hormones. Globulins transport ions, hormones and lipids assisting in immune function.
Proteins are electrically charged and they migrate towards the electrode when placed under an electric field. So, electrophoresis is a valuable tool to separate proteins in blood by exploiting their differential mobility in the electric field. The negatively charged proteins move to anode, a positive terminal and the rate of mobility of different serum proteins is determined by the balance between the driving force and the frictional force acting on them. The higher the rate of mobility, the closer the serum proteins move to the anode, Therefore, different serum proteins are separated in the electrophoresis.
In this experiment, cellulose acetate zonal electrophoresis was used as it can be applied to a wide variety of clinical electrophoresis including haemoglobin, serum protein and urine proteins with low molecular weight. This setup containing three main components which were DC power supply, electrophoresis chamber and supporting medium. The DC power supply provided a constant voltage and electric field producing a driving force to drive the protein serums and so separated five serum proteins into distinctive electrophoresis bands.
Analyzing of the electrophoresis band by Quantiscan gave a chromatogram so as to differentiate and identify the serum proteins. Barbital buffer was used to stabilize pH environment during the electrophoresis process. The buffer applied should be unreactive with serum proteins so as to give the accurate result. Tracking dye Bromophenol blue was used to monitor the process and as it is negatively charged at pH 8. 6, it migrated the same direction with the serum protein so the locations of the serum protein was stained. Materials and Methods Sample loading
First, the cellulose membrane was saturated with barbital buffer and the buffer saturated membrane was transferred to a filter paper. Then, a pencil line was drawn on the membrane. Human serum containing bromophenol blue dye was applied along the pencil line. Next, the membrane was placed into the electrophoretic tank and the machine was started. Staining of membrane First, the membrane was stained by Ponceau-S and then it was de-stained by acetic acid for three times. Next, water was removed in the membrane by absolute and cleaned by ethanol acetic acid.
The membrane was placed on the glass plate without bubbles trapped. Finally, the membrane was placed in the oven and analyzed by the Quantiscan. Results [pic] Figure 1 Photograph of the electrophoretic membrane |Serum Protein |*Color intensity |Color | |Serum Albumin |+++++ |Deep red | |? 1 Globulin | Not applicable | |? Globulin |+ |Pink | |? Globulin |++ |Pale red | |? Globulin |+++ |Red | Table 1 Table of analysis of the membrane The band color intensities represented the relative concentration of the serum proteins. The stronger the color intensity of the band of that serum protein, the higher concentration of that protein.
With above explanation that serum albumin had the highest concentration, followed by ? Globulin, ? Globulin and ? 2 Globulin. Figure 3 Chromatogram of the serum proteins The peak heights of the graph also represented the relative concentration of the serum proteins. The higher the peak, the higher the concentration of the serum proteins. This agreeed with the above explanation that the concentration of serum albumin was the highest followed by ? Globulin, ? Globulin and ? 2 Globulin in the membrane. [pic] Figure 4 Scanning of the electrophoretic membrane
There were 4 bands identified in the scanning and 4 peaks in the chromatogram. With reference to Y-axis, they were serum albumin, ? 2 Globulin, ? Globulin and ? Globulin accordingly. As different serum proteins have different sizes, they move at different rates under electrophoresis. The smaller the protein, the farer the get away from the starting point (200 in the Y-axis) due to the smaller resistance. Therefore, serum albumin had the smallest size and ? Globulin had the largest size accordingly. Discussion It was predicted that 5 peaks will be observed in the chromatogram.
However, only 4 peaks were shown in the result. Our result was not reflect the actual component of serum protein in human serum. It was believed that the peak for ? 1 Globulin was missing. Its peak should lie between the position of serum albumin and ? 2 globulin but it was not clearly observed. As the peak heights of the graph represented the relative concentration of the serum proteins. The higher the peak, the higher the concentration of the serum proteins. Therefore, this was probably because the concentration of ? 1 globulin was low so it cannot be detected and analyzed clearly.
Moreover, as different serum proteins have different sizes, they move at different rates under electrophoresis. The smaller the protein, the farer the get away from the starting point (200 in the Y-axis) due to the smaller resistance. Since the size of ? 1 globulin and ? 2 globulin are similar, they are not separated completely due to their similar mobility and so their peaks are fused. In addition, the use of low voltage in the electrophoresis, the serum proteins in sample cannot separate completely leading to the unobvious peak in ? globulin. The rate of mobility of a protein under an electric field is determined by the balance between the driving force and the resisting force acting on the molecule. The driving force depends on four factors which are the number and kind of charges per molecules, the degree of dissociation of the molecules in the buffer, the magnitude of the electrical field and the temperature. While the driving force is the force allowing the protein to migrate, the resisting force is an opposite force opposing the movement of the protein.
For the resisting force, it depends on another four factors which are the size and shape of the molecules, the viscosity of the medium, the ionic strength of the buffer and the solubility and adsorptive properties of the support medium. The higher the rate of mobility, the closer the serum proteins move to the anode, Therefore, different serum proteins are separated in the electrophoresis. The mobility of the proteins on the electrophoretic membrane can also be expressed by the equation. Molecular mobility (µ) = Net ionic charge (q) / frictional coefficient (f) The proof is given as followed:
Velocity of the molecule:v = Eq / f At constant electrical force:v = q / f Mobility (µ) as velocity per electrical unitµ = v / E Substitute by v = Eq / fµ = Eq / Ef = q / f Besides, there are additional factors affecting electrophoretic mobility. As the mobility is independent to the strength of electric field due to the constant power supply (100V in this experiment), only the velocity of the molecules is affected. According to the equation v = Eq/f, the velocity of the serum protein increase as the electric field. Also, Shielding of migrating molecules by buffer ions is also one of the factors.
Barbital buffer in this experiment was used as an electrolyte, a conducting solution. It acted as a buffer to stabilize the ionic environment and maintain pH in the electrophoresis so that the charge of the protein molecules would not change, i. e. , keeping the negative charge, during the process. Moreover, the electrophoretic mobility of the buffer counterions and the resolution of the gel will be affected by the choice of buffer. So, the buffer chosen should be unreactive and not modify or react with experimental serum proteins.
For electrophoresis, it is basically the interaction between migrating molecules and supporting medium. The motion of dispersed particles is relative to the electrolyte under the influence of a uniform electric field. In addition tracking dye Bromophenol blue was used in this experiment to monitor the process and indicate the stopping time of the experiment as it travels more rapidly than the serum proteins in the supporting medium. Since bromophenol blue carries negative charges at pH 8. 6, it migrates with the serum protein in the same direction and the locations of the specific serum protein is indicated.
There are different types of tracking dyes used in electrolysis for different purposes. For detection of proteins, silver staining is used. For detecting DNA, fluorescent dye or radioisotopes can be used. Major serum proteins are divided into two families which are albumin and globulins. There are four major types of globulins, each with specific properties and actions. For serum albumin, it carries steroid, fatty acids and thyroid hormones in blood and stabilizes extracellular fluid volume. It also acts as a major contributor of colloid osmotic pressure in plasma. For ? lobulin, including ? 1 globulin and ? 2 globulin, they inhibit certain blood proteases and some of them functioned as enzyme and carrier of compounds. For ? Globulin, it also acts as enzyme and carrier of compounds in the body, e. g, plasminogen and properdin. For ? Globulin, it is a kind of immunoglobulin which is a subclass of antibodies to boost patient’s immunity against disease. Serum albumin maintains the osmotic pressure of plasma so as to assist the transport of lipids and steroid hormones. Globulins transport ions, hormones and lipids assisting in immune function.
Various types of electrophoresis are used in diagnosis of diseases. For example, for Multiple Myeloma patients, a high serum protein, especially the concentration of globulins or immunoglobulin, is recorded in serum protein electrophoresis. If the globulin level is normal in established disease, protein electrophoresis of the blood and urine should be adopted to show the presence of a paraprotein band which is an abnormal immunoglobulin produced by the tumor clone. For the patient of Sickle Cell Anemia, Abnormal haemoglobin forms can be detected by haemoglobin electrophoresis which is a form of gel electrophoresis.
In which, various types of haemoglobin move at different speeds are observed. Sickle-cell haemoglobin and haemoglobin C with sickling can be identified from the experiment. Besides paper electrophoresis was used in this experiment, many others forms of electrophoresis were invented. Agarose gel electrophoresis is used to separate DNA fragments ranging from 50 base pair to several megabases. The distance between DNA bands of a given length is determined by the percent agarose in the gel.
As agarose gel is easily handled comparing to other matrices and gel setting is a physical rather than chemical change, samples are easily recovered. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, genetics and biotechnology to separate proteins according to their electrophoretic mobility. SDS is a detergent applied to a protein sample to linearize proteins. The binding of SDS to the polypeptide chain gives an even distribution of charge per unit mass, therefore, estimation of molecular weights of protein subunits can be completed by this electrophoresis.
Citation 1. Shim, J. ; P. Dutta and C. F. Ivory (2007). “Modeling and simulation of IEF in 2-D microgeometries”. Electrophoresis 28: 527–586. 2. Hunter, R. J. (1989). Foundations of Colloid Science. Oxford University Press. 3. Jacobs JM et al. (2005). “Utilizing human blood plasma for proteomic biomarker discovery”. Journal of Proteome Research 4 (4): 1073–1085. ———————– Serum albumin ? Globulin ?2 Globulin ? Globulin Serum albumin ?2 Globulin ?????????????????????????????????????????????????????????? ™????????? C??? Globulin ? Globulin
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