SERUM PROTEIN ELECTROPHORESIS (SPE)
|SOP Number: CH001
Author: Sue Galindo
Effective Date: August 25, 2009
This SOP applies to all staff who perform
clinical chemistry testing
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
CONSIDERATIONS/PERSONAL PROTECTIVE EQUIPMENT
- Use Standard Precautions when handling body fluids
- Refer to the Chemical Hygiene Plan for the proper
storage and use of chemicals
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.
- Electrophoresis apparatus including power supply and
- Drying oven set at 55°C
- Agarose film obtained from Beckman
- Clean forceps
- 1.0 uL transfer pipettes
- Graduated cylinder for measuring 95 mL
- Plastic trays for staining and destaining the gel
- Paper towels
- 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
- Glacial acetic acid (5% vol/vol)
- Amido black B10 stain prepared by dissolving 2 g in
5% vol/vol glacial acetic acid and make up to 1L
A normal serum control (Total Protein= 7.1
g/dL) is analyzed with each electrophoresis run with the
following ranges for each protein fraction:
- Set up the electrophoresis apparatus and turn on the
- Fill each of the electrophoresis chambers with 95 mL
of the electrophoresis buffer.
- 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.
- Fill each sample well with 1 uL of the serum to be
- Allow 2 minutes for the samples to soak evenly into
- 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.
- Connect to the power supply, processing it at 90 V
for 35 minutes.
- Following electrophoresis, remove the gel from the
cover, without inverting and place it on a flat paper
towel to drain.
- Transfer the gel to a container of amido black B10
stain for 10 minutes.
- Place the gel in a container containing 5% vol/vol
glacial acetic acid for 30 seconds.
- 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.
- Repeat step 10 two more times, each time using a
fresh 5% vol/vol glacial acetic acid for one minute
- Dry the gel in the oven at 55°C for 20 minutes
and then view the bands (Five distinct bands should be
- Use the densitometer to make a tracing of the bands
* units shown in g/dL
|16 days - 1 year
- Calculate the percentage of each protein band by
measuring the area under each curve
- Use the previously measured Total Protein result for
each sample to determine the amount in g/dL of each
protein fraction (g/dL) = % protein fraction ×
Total Protein (g/dL)
- Report each fraction in g/dL
- 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.
- 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.
- 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
- Loss of contact of the buffer with medium will
prevent migration of proteins.
- 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
- Bands may appear artifactually crescent-shaped and
is due to an overload of sample.
- Split peaks of albumin (bisalbuminemia) may be seen
in an inherited disorder.
- A transient or grossly widened albumin zone may be
due to albumin-bound medications.
- 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.
- In addition to Amido black 10B, Coomassie brilliant
blue or Ponceau S may be used to stain the protein
- 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
- If centrifugation occurs before complete clotting of
the whole blood, fibrinogen may be present and will
cause an artifactual increase in the β globulin
- Burtis, Carl A et al. Tietz Fundamentals of Clinical
Chemistry, 6th ed. Saunders: St Louis, Missouri, 2008.
- Naser, Najih. Clinical Chemistry Laboratory Manual.
Mosby: St Louis, Missouri, 1998.