Serum protein electrophoresis is a simple, affordable method of separating blood proteins based on electrical charge, size and shape. In the blood serum there are two major categories of proteins: albumin and globulin. Albumin has the largest concentration and globulines are in a smaller amount, but they are the basis for electrophoresis. There are 5 categories of globulins: alpha-1, alpha-2, beta-1, beta-2 and gamma.
The patient’s serum is placed on a special medium (e.g., cellulose, agar) and then an electric charge is applied for a period of time when the proteins will migrate from the negative pole to the positive one. Albumin migrates and reaches the positive pole and gamma globulins will remain at the negative pole, resulting in the fractions specific to each protein.
With the help of the staining step, the fractions are highlighted and can be interpreted by means of readers that measure optical density. The results are expressed for each type of protein in part and expressed as: normal, low, increased.
Work on a clean and flat surface, with all the necessary materials at hand. Carefully read the instructions.
PREPARATION OF SOLUTIONS
In a 100 ml cylinder, add 10 ml buffer from the original bottle and 90 ml of distilled water and mix. It is very important to respect the 1: 9 ratio!
The solution is ready to work
In a 100 ml cylinder, add 10 ml of decolorizing solution and 90 ml of distilled water and mixe. It is very important to respect the 1: 9 ratio!
The solution is ready to work
PREPARATION OF SAMPLES
The serum may be fresh or stored at 2-8°C for up to 72 hours. Lipemic or hemolysed serum is not working.
Dilute serum with buffer: 20μl serum + 120μl buffer in an eppendorf vial.
Open the gel plate on which the patient’s diluted serum will be applied and place on a support (white sheet of paper).
Apply the filter paper to the foil at the points where the samples will be applied to remove excess moisture for a few seconds.
Raise the filter paper and immediately place the applicator on which the samples are pipetted, with great care to make complete contact with the gel.
Prior to applying each sample, the serum buffer mixture will shake.
Pipette 3.5μl of dilute serum along each well and allow to absorb completely for 5-8 minutes, not more than 10 minutes, excess serum will be removed by means of a filter paper and then remove the applicator.
Pipette 35 ml of buffer solution into each side of the cuvette (+/-) (buffer change is recommended after each migration).
Put the foil in the cuvette so that you adhere + on the foil with + on the cuvette and – on the foil with – on the cuvette and cover the cuvette.
Fix the parameters specified in the protocol and start migration.
After the migration is complete, remove the foil immediately and insert it into the gel fixation bath for up to 7-10 minutes.
After the fixing step, the foil is removed from the solution bath and placed in the oven at a minimum of 90oC for about 20 minutes.
After drying the foil it is put in the dye bath 6-8 minutes.
Remove the staining solution from the bath and make a destaining solution over the foil. It fades into 2-3 successive baths. The foil is left to dry completely (possibly at the oven at 70-80oC).