Electrophoresis

notes on electrophoresis, useful for postgraduate medical and paramedical students

1. 1
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics



ELECTROPHORESIS

2. 2
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
OVERVIEW

1. History
2. Zonal electrophoresis
 Principle
 Method
3. Buffer
4. Support media
5. Ancillary techniques before performing electrophoresis
 Precipitation
 Column separation
- Gel filteration
- Ion exchange chromatography (anion exchange, cation exchange)
- Hydrophobic chromatography
- Affinity chromatography
- Capillary electrophoresis
6. Summary of workflow
7. Applications
a. Serum protein electrophoresis
b. CSF electrophoresis
c. Urine electrophoresis
d. Hemoglobin electrophoresis

3. 3
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
* HISTORY

1. The technique of electrophoresis was introduced by Tiselius.
2. His technique is known as MOVING BOUNDARY ELECTROPHORESIS / FRONTAL
ELECTROPHORESIS
3. He separated proteins dissolved in electrolyte solution by application of electric current
through U shaped quartz tube that held the solution.
4. At ph 7.6, the solution got separated into four distinct protein fractions, albumin, α, β
and γ. These separated proteins were identified by checking the change in refractive
index across the solution.
5. As there was no solid support to the medium, these separated proteins did not form
distinct zones.

4. 4
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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* ZONAL ELECTROPHORESIS

1. In Teselius’ technique, the proteins cound not form distinct bands
2. This problem was solved by introducing an anticonvection solid support medium – like
filter paper.
3. Now on applying electric current, the proteins separated into discrete bands at ph 8.6
4. The α band got further split into α1 and α2.
5. Other solid media that are frequently used nowadays include cellulose acetate
membrane, agarose gel, starch gel and polyacrideamide gel.



Anode Cathode

Principle:

1. The proteins to be separated are applied at anode.
2. The support medium has negative charge – the electromotive force (determined by
charge of proteins and pH) tends to move the support medium towards anode (positive
pole) – but support medium cannot move.
3. The buffer ions have positive charge – they tend to move towards cathode.
4. This net result is flow of buffer towards cathode – this force called electro osmotic force
(endosmosis).
5. The buffer ions carry with them water molecules and protein molecules. Proteins here
move not only on the basis of their charge, but also determined by their molecular
weight.
6. This explains the movement of slightly negatively charged γ globulins towards the
cathode.
7. The actual distance traveled by the protein is dependent on these two forces described
above.

5. 5
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
Method:

1. The sample to be separated is soaked into a gel.
2. Each end of the gel is then inserted into separate buffer chambers in which electrodes
are mounted.
3. An electric current is then passed through the gel for a period of, about 30 minutes
usually.
4. After this the gel is treated with a mild fixative such as acetic acid.
5. This fixative precipitates the proteins at the positions on the gel to which they have
migrated to.
6. They are then stained and the gel is dried.
7. The protein patterns can now be visually inspected for qualitiative identification of
abnormal proteins.
8. To find out the quantity of each type of protein present, densitometric scanners are
used, they generate tracings and quantitate relative percentages of proteins in each
fraction.
9. These percentages when multiplied by total proteins can give an idea about the
concentration of protein in each fraction.
Eg. If total protein is 9 gm/dl and albumin fraction obtained is 50% - = 4.5 gm/dl
(9 x 50/100) is the albumin concentration
10. On varying the buffer media / solid media – different applications of electrophoresis can
be developed.

6. 6
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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* Varying buffer concentration and its application

1. If endosmosis is set at a high, this maximizes cathodal movement.
2. Immunoglobulins can be separated optimally and this helps in detecting oligoclonal
bands in CSF

7. 7
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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* Varying solid media and its applications

PAGE – Polyacrylamide Gel Electrophoresis

1. Polyacrylamide is an inert support medium
2. It is useful for standard separation of native proteins based on charge

8. 8
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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SDS-PAGE – Sodium dodecyl sulphate Polyacrylamide gel electrophoresis

1. SDS denatures proteins according to their molecular weight
2. SDS-PAGE is very powerful for resolving proteins and separating them into subunits
according to their molecular weight
3. Very useful for research purposes

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Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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TWO DIMENSIONAL ELECTROPHORESIS (2D ELECTROPHORESIS)

2. Standard separation of proteins is done in one direction
3. This is followed by SDS-PAGE in perpendicular direction
4. This results in hundreds of identifiable protein peaks

10. 10
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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ISOELECTRIC FOCUSSING

1. Gives superior resolution to closely migrating proteins
2. Here the gel used contains a gradient of pH established by using ampholytes
3. As each protein reaches the gel location where the pH equals its own, the net charge
becomes zero
4. It cannot move further
5. Thus proteins move here strictly on the basis of their pH

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Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
* ANCILLARY TECHNIQUES

PRECIPITATION

1. The sample to be tested usually contains a lot of undesired proteins.
2. These undesired proteins can be removed by precipitation so that a single minor protein
can be isolated by electrophoresis.
3. First the value of total proteins is found out
4. Then Sodium sulfate, sodium sulfite, ammonium sulfate or methanol is used to
precipitate globulins.
5. Protein estimation from the remaining solution after precipitation can give an idea
about the albumin concentration – electrophoresis can give an idea about the types of
proteins apart from globulins.
6. globulin = total protein – albumin
7. estimation of albumin : globulin ratio is useful in various disorders

Normal :
A:G ratio >1:5 (1-3)

Decreased A:G ratio Increased A:G ratio
Decreased albumin
Malnutrition
Malabsorption
Liver failure
Proteinuria
Accumulation of ascetic fluid
Enteropathy

Increased globulins
Acute or chronic infections
Myelomas
************

12. 12
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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COLUMN SEPARATION

(1) Gel filteration:

1. Media such as agarose beads/sephadex is used
2. They have beads in them with pores of various sizes
3. Largest proteins cannot pass through pores in the beads – they pass through spaces in
beads and are eluted first
4. Medium sized proteins pass through medium sized pores and appear second.
5. Small sized proteins pass through small sized pores and are eluted last.

13. 13
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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(2) Ion Exchange Chromatgraphy

1. This is of two types – Anion exchange and Cation exchange chromatography
2. This technique uses charged particles which are placed in the column.
3. When proteins having the opposite charge as the column pass through it, they are
retained for a longer time, neutral elute the second while same charged particles elute
first.



ANION EXCHANGE CHROMATOGRAPHY

1. Media such as DEAE or QAE are used with have net positive charge.
2. Albumin, α and some β globulins which are anions stick to positively charged matrix.
3. γ globulins that have no charge are eluted faster.

CATION EXCHANGE CHROMATOGRAPHY

1. Media such as carboxymethylcellulose are used which have a negative charge.
2. Positively charged cations stick to the matrix
3. Albumin is eluted first here

14. 14
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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(3) Hydrophobic chromatography

1. In this technique, support medium interacts with proteins according to their
hydrophobic nature.
2. Here ‘high salts’ and ‘low salts’ are used
3. high salts structure water around themselves and expose the hydrophobic ends of
proteins
4. these hydrophobic ends get attracted toward the medium and stick to it
5. according to hydrophobic nature of proteins, they stick or are eluted
6. ‘low salts’ are used to elute as they donot / expose less number of hydrophobic ends.

15. 15
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
(4) Affinity chromatography

1. This is based on specific binding between a protein of interest and another protein (can
be its antibody/antigen) bound to the solid medium.
2. Bound protein can then be eluted in its purified form.
3. examples
a. to separate FVII-vWF – column is incorporated with monoclonal antibody to
vWF
b. Igs can be separated by using Staphylococcal protein A in gel matrix
c. Attaching dihydroxyboronate to medium helps separate glycosylated
hemoglobin
d. To separate digoxin – digibind is used
CroFab – crotalid snake venom

16. 16
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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(5) Capillary electrophoresis

1. This procedure is similar to HPLC in that a column is employed where separation of
molecules is done on the basis of size/hydrophobicity or strereospecificity.
2. Applicable to large molecules such as DNA.

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Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
* SUMMARY OF WORK FLOW

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Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
Contact: [email protected] Facebook: facebook.com/pathologybasics
* APPLICATIONS

(1) SERUM PROTEIN ELECTROPHORESIS

Following patterns are observed in normal conditions and various disorders on
electrophoresis.

Normal:




Cirrhosis of liver:

When liver function is sufficiently diminished, protein synthesizing capacity is
compromised and concentrations of albumin and proteins in the alpha and beta bands are
decreased. An additional common finding is beta-gamma bridging due to increased IgA.








Beta-gamma bridging



Alb α1 α2 β γ

19. 19
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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The Nephrotic Syndrome --
1. Renal disease involving the glomeruli is always associated with increased urinary protein
loss. When protein loss is greater than 3-4 g/day, the protein synthesizing capacity of
the liver is exceeded and hypoproteinemia, accompanied by anasarca, develops to
cause the nephrotic syndrome.
2. The massive urine protein loss is due to increased permeability of glomeruli to protein.
The permeability increase may be minimal so that only albumin and other smaller
molecular weight proteins are selectively filtered (selective nephrosis, as in Minimal
Change Disease) or may be greater so that larger proteins are also filtered (nonselective
nephrosis, as in membranous golmerulonephritis) as is the case in the example shown.
3. Alpha-2-macroglobulin is sufficiently large so that it is not filtered and increased
synthesis (from the general hepatic protein synthesis) causes its accumulation.
4. Lipoproteins are also sufficiently large to accumulate and hyperlipidemia is a
characteristic of the nephrotic syndrome, although lipoproteins are not stained with the
protein stain used in visualizing proteins.

Normal
Abnormal







Alb α1 α2 β γ



Alpha-1-Antitrypsin Deficiency:
A genetic defect causes a deficiency of alpha-1-antitrypsin. The antiprotease deficiency
results in a propensity to develop emphysema. Since alpha-1-antitrypsin is the major
component of the alpha-1 band, deficiency is suggested by a reduced alpha-1 band. Deficiency is
confirmed by specific immunochemical quantification.



Normal
Abnormal







Alb α1 α2 β γ

20. 20
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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Acute Inflammation
The alpha-1 and alpha-2 bands are increased during the inflammatory response from
increased hepatic synthesis of acute phase reactant proteins.

Normal

Abnormal






Alb α1 α2 β γ



Chronic Inflammation --

Immunoglobulin synthesis by antigen activated B lymphocytes transformed to plasma
cells is demonstrated by the increased polyclonal gamma band.


Normal
Abnormal








Alb α1 α2 β γ

21. 21
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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Immunoglobulin Deficiency --

Deficient immunoglobulin synthesis is revealed by a markedly diminished gamma band.
Effected individuals are prone to recurrent infection














Monoclonal Gammopathy --

1. An unusually sharp band in the gamma region strongly suggests the presence of a
homogeneous immunoglobulin and, thus, the malignant proliferation of plasma cells
from a single cell (multiple myeloma) in contrast to the broad, heterogeneous, or
polyclonal, gamma band as exhibited above in chronic inflammation from
immunoglobulin synthesis by many different clones of plasma cells.
2. Homogeneous immunoglobulins are also found in Waldenstrom's macroglobulinemia
(where the sharp gamma band is always IgM). Specimens which exhibit a narrow
gamma band are further examined by immunofixation electrophoresis as described
below











Alb α1 α2 β γ





(2) CSF ELECTROPHORESIS

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Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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1. The diagnosis of Multiple sclerosis is ultimately a clinical one based on neurologic
history and physical examination.
2. Nevertheless, advanced laboratory results such as elevated IgG indices and presence of
oligoclonal bands of IgG or Kappa /lambda light chains on CSF electrophoresis, as well as
neuroimaging techniques have proven to be invaluable in diagnosis of MS.

Agarose gel electrophoresis

1. Concentrated CSF is used as sample
2. Shows discrete bands of IgG – the oligoclonal bands
3. Presence of two or more bands is necessary for diagnosis
4. False positive results are obtained in SSPE, CNS infections like neurosyphilis,
Crypotococcal meningitis, GBS, transverse myelitis, burkitt’s lymphoma etc.
5. Sensitivity is less than other methods of electrophoresis

Staining

Commassie brilliant blue/paragon violet/silver staining

1. silver staining is more sensitive then CBB
2. however these techniques must be simultaneously carried out on patients serum also
because polyclonal gammopathy can lead to diffusion of immunoglobulin into CSF and
lead to false positive results. This is seen in case of liver diseases, SLE, RA and chronic
granulomatous diseases.

ImmunoFixation Electrophoresis

1. Immunofixation electrophoresis (IFE) is used to demonstrate that a narrow gamma band
is due to a homogeneous immunoglobulin.
2. In the illustration below, 6 replicates of the specimen are loaded on to separate lanes of
an IFE gel.
3. Following electrophoresis, the protein in each lane is stained differently.
4. The first lane (SP) is stained for total protein.
5. The protein in each of the other lanes is "stained" with specific antisera for
immunoglobulin heavy and light chains, respectively, as illustrated in the figure.
6. The finding of the preponderance of only one light chain associated with a
predominantly staining heavy chain confirms the molecular homogeneity of the
immunoglobulin and also provides identification.
7. IFE identifies the narrow band as monoclonal IgG, lambda.
8. Sometimes malignant plasma cells synthesize excess light chains and less frequently
only light chains are synthesized. Almost never is excess heavy chain or only heavy chain
synthesized. Excess free lambda light chain is exhibited in the illustration.

23. 23
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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a. Lymphocytic pleocytosis and 2 plasma cells in the CSF cytospin preparation.
b. 4b. Isoelectric focusing/immunofixation revealed CSF-restricted oligoclonal IgG
bands as well as identical paraprotein bands in CSF and serum. Anode is on the
left.
c. 4c. Serum immunofixation electrophoresis revealed the presence of IgGκ
monoclonal band (arrow). Anode is at the top. ELP, electrophoresis (general
protein stain); G, A, M, K, L: immunofixation for γ, α, μ, κ and λ chain,
respectively.
d. 4 d. Isoelectric focusing/affinity-mediated immunoblotting analysis: Paraprotein
bands did not react with Borrelia antigen but partly obscured CSF-restricted
oligoclonal IgG pattern. Paired CSF and serum samples tested for (from left to
right) "total" IgG, IgGκ, IgGλ; Anti-Borrelia IgG, IgGκ, IgGλ. Anode is at the top.

24. 24
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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(3) URINE ELECTROPHORESIS

1. The main reason for performing urine protein electrophoresis is to find a light chain
myeloma producing an excess of free light chains (Bence Jones protein), an important
part of a myeloma screen.
2. A band in the urine protein electropherogram may also result from an intact monoclonal
immunoglobulin, especially if the patient has poor renal function.
3. Immunofixation is important in defining the nature of the band and in distinguishing
between Bence Jones protein and an intact monoclonal protein originating from the
serum.
4. From the urine electropherogram we can also tell if the proteinuria is of glomerular
origin with a predominance of albumin, or if it has tubular components with excretion of
smaller molecular weight proteins such as retinol binding protein and alpha-1
microglobulin.
5. Fragmented albumin in urine is occasionally seen but is of unknown significance.
6. Historically, urine has been concentrated by either removal of water from the specimen
leaving the proteins in higher concentration, or by centrifugation whereby the proteins
are spun away from the majority of the water.
7. Demonstration of the protein components of urine from concentrated specimens was
originally performed on cellulose acetate and later on agarose and high resolution
agarose gel.

25. 25
Notes on electrophoresis.. By Dr. Ashish V. Jawarkar
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(4) HEMOGLOBIN ELECTROPHORESIS

Rationale:

1. Haemoglobin (Hb) abnormalities are the most frequent genetic disease, affecting
approximately 7 per cent of the world population.
2. Today, abnormal Hbs are generally discovered during a systematic study performed
within programmes for prevention of thalassaemias or sickle cell disease.
3. In several regions (India, Turkey, Irak, Iran, Gaza strip, Saudi Arabia, Cyprus, etc.), these
are found during a premarital screening. In other regions, like west Europeans countries,
the research for the main Hb disorders is often limited to populations at risk. It is done
either as preconceptional or neonatal screening programmes.

Methods and patterns:

1. Hb S [β6 Glu>Val], the first abnormal Hb described, was discovered in 1949 by Pauling et
al using moving boundary electrophoresis.
2. Zone electrophoresis performed on cellulose acetate strips (CAE) is still used in many
clinical laboratories.
3. This technique has a resolution lower than that of IEF, but because of its simplicity
remains among the more popular methods used in Hb screening. In this technique, the
Hb molecules are separated at alkaline pH. Under these conditions all Hbs have a
negative charge and migrate towards the cathode. Hb S, which has an additional
positive charge compared to Hb A migrates more slowly.
4. In IEF, a pH gradient is established by carrier ampholytes submitted to an electric
current and the Hb molecules migrate across this gradient until they reach the position
where their net charge is zero (isoelectric point). The molecules will then concentrate in
a sharp band as illustrated in Fig.
5. This technique allows separating molecules with isoelectric points differing only by 0.02
pH unit. Hb D-Punjab [β121 Glu>Gln], not detectable from Hb S by CAE, could easily be
recognized by this technique. The disadvantage of this method is its relatively high cost
and the requirement for a well-experienced laboratory staff.
6. This technique can help in diagnosing various hemoglobinopathies.