LIFS 2720 – Introductory Biochemical Laboratory
Lab Report 4
Serum Electrophoresis Using Cellulose Acetate
CHAN Kin Yan
Group No. 9
Date of Experiment: 1st March 2012
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 γ Globulin, β Globulin and α2 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 γ 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 α1 Globulin was missing as it had low concentration and similar size with α2 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.
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 serum. 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...
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