SERUM PROTEIN ELECTROPHORESIS (SPE)


SOP Number: CH001
Author: Sue Galindo
Version: 1
Effective Date: August 25, 2009

SCOPE
This SOP applies to all staff who perform clinical chemistry testing
TEST PRINCIPLE/CLINICAL SIGNIFICANCE
A charged particle placed in an electrical field migrates towards the anode or cathode depending on the net charge carried by the particle. Serum proteins carry a negative charge at pH 8.6 and move toward the anode. Serum is placed into a sample trough within agarose gel in an alkaline buffer. A standardized voltage is applied and upon staining, five distinct protein bands (albumin, α1, α2, β, and γ) are seen. The relative migration rate of proteins is based on ionic charge. The membrane is then run through a densitometer and the light absorbance by the protein bands is recorded. The absorbance of light is proportional to the relative protein concentration. The protein concentration of each band in g/dL is then calculated.
SAFETY CONSIDERATIONS/PERSONAL PROTECTIVE EQUIPMENT
  1. Use Standard Precautions when handling body fluids
  2. Refer to the Chemical Hygiene Plan for the proper storage and use of chemicals
SPECIMEN TYPE
Fresh serum or serum stored up to five days at 2 to 8°C. Frozen serum can be used if gently thawed and thoroughly mixed prior to use.
REQUIRED REAGENTS/SUPPLIES/EQUIPMENT
  1. Electrophoresis apparatus including power supply and densitometer
  2. Drying oven set at 55°C
  3. Agarose film obtained from Beckman
  4. Clean forceps
  5. 1.0 uL transfer pipettes
  6. Graduated cylinder for measuring 95 mL
  7. Plastic trays for staining and destaining the gel
  8. Paper towels
  9. Barbital buffer, pH 8.6 prepared by dissolving 15.40 g sodium diethyl barbiturate and 2.76 g barbituric acid in water and make up to 1L volume
  10. Glacial acetic acid (5% vol/vol)
  11. Amido black B10 stain prepared by dissolving 2 g in 5% vol/vol glacial acetic acid and make up to 1L
QUALITY CONTROL
A normal serum control (Total Protein= 7.1 g/dL) is analyzed with each electrophoresis run with the following ranges for each protein fraction:


g/dL
Albumin 3.5-5.2
α1-Globulin 0.2-0.4
α2-Globulin 0.4-0.8
β-Globulin 0.5-1.1
γ-Globulin 0.6-1.3
PROCEDURE
  1. Set up the electrophoresis apparatus and turn on the drying oven.
  2. Fill each of the electrophoresis chambers with 95 mL of the electrophoresis buffer.
  3. Remove the agarose gel from its package. Handle the gel by its edges using the forceps and place it gel side up on a clean flat countertop.
  4. Fill each sample well with 1 uL of the serum to be tested.
  5. Allow 2 minutes for the samples to soak evenly into the wells.
  6. Insert the agarose film into the electrophoresis cell cover, agarose side up, matching the (+) and (-) signs on the cell cover with those on the gel.
  7. Connect to the power supply, processing it at 90 V for 35 minutes.
  8. Following electrophoresis, remove the gel from the cover, without inverting and place it on a flat paper towel to drain.
  9. Transfer the gel to a container of amido black B10 stain for 10 minutes.
  10. Place the gel in a container containing 5% vol/vol glacial acetic acid for 30 seconds.
  11. Remove the gel from the tray, wipe away any excess fluid, and then incubate the gel in the drying oven at 55°C for 15 minutes.
  12. Repeat step 10 two more times, each time using a fresh 5% vol/vol glacial acetic acid for one minute each time.
  13. Dry the gel in the oven at 55°C for 20 minutes and then view the bands (Five distinct bands should be observed)
  14. Use the densitometer to make a tracing of the bands
REFERENCE INTERVALS

age
Albumin α1-Globulin
α2-Globulin
β-Globulin γ-Globulin
0-15 days
3.0-3.9
0.1-0.3
0.3-0.6
0.4-0.6
0.7-1.4
16 days - 1 year
2.2-4.8
0.1-0.3
0.5-0.9
0.5-0.9
0.5-1.3
1-16 years
3.6-5.2
0.1-0.4
0.5-1.2
0.5-1.1
0.5-1.7
>16 years
3.9-5.1
0.2-0.4
0.4-0.8
0.5-1.0
0.6-1.2
* units shown in g/dL

RESULTS / INTERPRETATION
  1. Calculate the percentage of each protein band by measuring the area under each curve
  2. Use the previously measured Total Protein result for each sample to determine the amount in g/dL of each protein fraction:
    protein fraction (g/dL) = % protein fraction × Total Protein (g/dL)
  3. Report each fraction in g/dL
PROCEDURE NOTES
  1. Urine and CSF can also be tested but must be concentrated to a protein content of at least five g/dL using a concentration filter device.
  2. Examine all materials for signs of deterioration or contamination. Bacterial growth impedes separation of proteins which can be overcome by using fresh buffer and keeping it refrigerated between uses. Do not use if the materials have exceeded the expiration or if there are signs of deterioration.
  3. Although γ-Globulins are negatively charged at pH 8.6, in some cases the buffer flow is strong enough to carry these molecules against the electrical force. This phenomenon is known as endosmosis or electro-osmosis.
  4. Loss of contact of the buffer with medium will prevent migration of proteins.
  5. Each band in the stained protein pattern should be uniformly colored; that is, no holes should appear within an individual band. Such a doughnut-like appearance occurs when the protein is present in too high a concentration, thus exceeding the complexing ability of the stain. To overcome this problem, dilute elevated specimens before repeating the electrophoresis.
  6. Bands may appear artifactually crescent-shaped and is due to an overload of sample.
  7. Split peaks of albumin (bisalbuminemia) may be seen in an inherited disorder.
  8. A transient or grossly widened albumin zone may be due to albumin-bound medications.
  9. Cellulose acetate, starch gel, and acrylamide are also used as support mediums in some electrophoresis systems. The pore size of the agarose gel and cellulose acetate is large enough that the protein molecules are able to move freely through the media with a resolution of between 5 to 7 fractions. Because the pore size of starch gel and acrylamide is somewhat smaller, the resolution of approximately 20 fractions is possible with this type of medium.
  10. In addition to Amido black 10B, Coomassie brilliant blue or Ponceau S may be used to stain the protein bands.
LIMITATIONS
  1. Hemolysis in the serum causes an increase in the β globulin fraction due to free hemoglobin or in the α 2 globulin fraction due to haptoglobin-hemoglobin complexes.
  2. If centrifugation occurs before complete clotting of the whole blood, fibrinogen may be present and will cause an artifactual increase in the β globulin fraction.
REFERENCES
  1. Burtis, Carl A et al. Tietz Fundamentals of Clinical Chemistry, 6th ed. Saunders: St Louis, Missouri, 2008.
  2. Naser, Najih. Clinical Chemistry Laboratory Manual. Mosby: St Louis, Missouri, 1998.
APPROVAL
APPROVED BY
Dr. QC
SIGNATURE DATE
6/24/2009
REVIEWED BY
Susan Galindo
SIGNATURE DATE
8/25/2011