Beta globulins include low-density substances involved in fat transport (lipoproteins), iron transport (transferring), and blood clotting (plasminogen and complement).
Gamma globulins are all antibodies—proteins produced by the immune system in response to infection, allergic reactions, and organ transplants. If serum protein electrophoresis has demonstrated a significant rise at the gamma-globulin level, immunoelectrophoresis is done to identify the specific globulin that is involved.(1)
All proteins have an electrical charge. The SPEP test is designed to make use of this characteristic. There is some difference in method, but the sample is placed in or on a special medium and an electric current is applied to this medium. The protein particles move through the medium according to the strength of their electrical charges, forming bands or zones. These bands are then measured, by elution, solution or scanning, and can be identified and associated with specific diseases.
The purpose of this experiment is to establish the time required to adequately separate the various serum protein fractions using cellulose acetate as a support medium.
Method :
- Obtained the prepared Barbitol (Sodium Barbiturate) Buffer and poured approximately 900 mL into the electrophoresis chamber, the leveled the buffer by tilting the chamber.
- Connected the power supply, but did not turn it on.
- Using care, we labeled six cellulose acetate strips, marking each with a pencil line 1 1/4 inches from one end. These were then soaked in about 150 mL of the buffer in a staining tray for 5 minutes.
- The sample applicator was washed with soap solution, tap water, DI water and then acetone, in turn. The acetone wash was disposed of in the organic waster container.
- Under the fume hood a 5% solution of aqueous acetic acid was prepared, using 50 mL of glacial acetic acid added to approximately 500 mL DI water in a 1 L volumetric flask. The acid was added to the water, which was then topped up to the mark with more DI water. This was capped and inverted 24 times to mix.
- The acetate strips were removed one at a time from their bath and blotted on absorbent paper, but not allowed to dry. The strips were then draped across the supports in the electrophoresis chamber and held into place by magnets in such a way so that only the ends of the strips touched the buffer solution.
- Using the sample applicator a sample of 1L Test SeraChem level 1 [(human)(Lot N1054503 exp July’97)] was applied to each strip at the pencil mark on the left side. The chamber was then covered with the plastic lid.
- The power was turned on and voltage set to 250 volts.
- The buffer used to soak the acetate strips was poured back into the bottle for reuse. The tray was rinsed and about 150 mL of Ponceau-S stain was put in the tray.
- The 5% aqueous acetic acid solution was evenly divided into 3 rinse trays.
- Using a stopwatch each strip in the electrophoresis chamber was removed one by one at five-minute intervals. Each strip was placed in the stain tray until just before the next strip was due to be removed from the chamber. Then the stained strip was moved through each of the rinse trays as the strips were removed. Finally, the strips were all removed, stained, rinsed, and put on a blotter to dry.
- The Barbitol Buffer was put back into the storage bottle for re-use. The Ponceau-S stain was put back into its bottle for re-use.
- Once the strips were all dry, we tried to identify the one that appeared to have the best separation of fractions and identified as many as possible.
Results :
The six cellulose acetate strips all showed a movement from the marked end, where the sample was applied, to between 1 inch and about 2 inches further down the strip.
Strips 1, 4, 5 & 6 showed the least differentiation, and while #2 showed better definition, #3 showed the most fractions. #3 was removed from the buffer solution at the 15-minute mark.
Discussion :
According to the strips (shown attached), the best separation was achieved after 15 minutes. This is indicated by the identification of five different bands. Less than 15 minutes had some good separation, while after 15 minutes had very little separation.
While the separation of strip #3 was the best of the six, it still was not as clear as we would have expected. This could be due to the age of the electrophoresis chamber, being at least 30 years old; it would be safe to assume that modern methods are much finer in their definition of separation. There is also the fact that we had never used this type of equipment before and could have done with a little more practice in the application of the sample. Another factor that could have made a difference would be the amount of the voltage used. While the minimum called for was 220 volts, we used 250 volts, but recommended range went up to 300 volts.
There are several factors which can affect the electrophoretic mobility : the strength of the electrical field, the size of the protein molecules, their net charge, the nature of the conducting fluid (pH) and the nature and type of support (paper, agar, acrylamide gel, agarose or cellulose polyacetate). As electrophoresis is a diagnostic tool for specific diseases, the sample can also cause differing mobilities as the amounts of the proteins involved increase or decrease. For example, a decrease in albumin with a rise in the apha2 globulin usually indicates an acute reaction of the type that occurs in infections, burns, stress, or heart attack. On the other hand, a slight decrease in albumin, with a slight increase in gamma globulin, and a normal alpha2 globulin is more indicative of a chronic inflammatory condition, as might be seen is cirrhosis of the liver. (1)
Reference :
- FLORES, JANIS O. “Protein electrophoresis”
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November 3, 2002