Electrophoresis

EP refers to the migration of changed

solutes or particles in a liquid medium
under the influence of an electrical field

OR

EP is separation of charged compounds

based on their electrical charge
Zone E.P describes the migration of
charged macromolecules in a porous
supporting medium, such a cellulose
paper, cellulose acetate sheets and
agarose gel film.
 PRINCIPLE OF EP
When a voltage is applied to a salt solution (>>
sod.chloride), an electrical currents produced
by flow of ions : cations toward the cathode and
anions toward the anode. Conductivity of sol.
increases with its total ionic concentration. The
greater the net charges of compound,the faster
it moves through the solution toward the
oppositely charge electrode.The net charge of a
compound, in turn, depend on the solution pH.
The rate of migration are dependent on :

1. Net electrical charge of the molecule

2. Size and shape of the molecule

3. Electrical field strength

4. Properties of the supporting medium

5. The temperature of operation

The most common application of EP :
1. Serum proteins
2. Hemoglobins
3. Isoenzymes  CK, LD and AP
Types of EP

 Agarose Gel EP (AGE)

 Celluler Acetate EP (CAE)
 Polyacrylamide Gel EP (PAGE)
 Starch EP
 Isoelectric Focusing (IEF)
 Type of EP
Agarose Gel EP (AGE)
successfully applied to analysis of serum
proteins, Hb variants, LDH isoenzymes,
lipoprotein fractions & other substances
Advantages :
* Lower affinity for proteins
* Native clarity after drying
* Requires small sample size
* Short time (30-90 min)
Cellulose Acetate E.P (CAE)
Advantages :
* The speed of separation (20 min to
1 hour)
* The ability to store the transparent
membranes for long periods
Isoelectric Focusing (IEF)
Separates amphoteric compounds
by virtue of migration in a medium
possessing a stable pH gradient
Polyacrylamide Gel E.P (PAGE
Advantages :
PAGE may yield 20 or more fractions
(AGE and CAE : 5 –7 zones)  widely
use in the study of individual proteins,
genetic variants and isoenzymes
Two Dimensional E.P (2D E.P)
uses a charge-dependent IEF in first
dimension and MW-dependent E.P in
the second
Capillary E.P
methods based on light absorption,
fluorescence and electrochemical,
radiometric and mass spectrometric
techniques  possible detection of
as little as 10-20 of substance in the
injected volume.
Limitation and Errors in Routine E.P

Wick Flow : the heat that is producing

during E.Phoretic causes evaporation of
solvent from E.P support
Electroendosmosis
Buffers
Stain solution
Sampling
Sample application
 Buffer

Carry the applied current

Fix the pH when EP is carried out
Barbital, Tris-boric acid EDTA
Effect between buffer and protein is
minimal
 Protein Stains Used in EP
Procedures

 Amido Black, Ponceau S, Bromophenol

Blue
 Silver Nitrate
 PAGE IEF: Coomassie Brilliant Blue
Manfaat Klinik

 Penyakit inflamasi
 Keganasan
 Sindroma Nefrotik
 Liver diseases (kronik)
 Status Nutrisi
 Oedem dengan kausa belum jelas
Protein Total Meningkat

 Dehidrasi dan hemokonsentrasi

 Penyakit hati
 MM atau Gamopati lain
 Wadenstorm makroglobulinemia
 Sarkoidosis
 Penyakit kolagen
 Infeksi/peradangan kronik
Protein Total Menurun

 Starvasi dan Malabsorbsi

 Severe liver disease
 Alkoholisme
 Sindrom nefrotik
 Luka bakar
 Perdarahan hebat.
Albumin Serum Meningkat

 Pasca Infus albumin

 Dehidrasi (peningkatan Hemoglobin dan
Hematokrit)
 Albumin Serum Menurun
 Gangguan sintesa albumin
Penyakit hati, alkoholisme, malabsorbsi,
starvasi, penyakit crohns
 Kehilangan albumin
Sindrome nefrotik, luka bakar derajat III
 Status nutrisi yang jelek, Albumin-Globulin
Ratio yang rendah
 Penyakit kolagen, keradangan kronik, peny.
Hati, makroglobulinuria, infeksi berat,
kekeksia, luka bakar dan kolitis ulserosa
SPE Beberapa Penyakit Yang
Umum
 Sirosis hati (Alb. Rendah, Gamma
Globulin Meningkat)
 SN (Alb. rendah, Alfa 2 tinggi, Beta
turun, Gamma turun)
 MM ( Alb. Rendah, Gamma meningkat
tinggi dan sempit)
SPE Beberapa Peny. Umum

 Penyakit Autoimun (Albumin rendah,

Gamma meningkat)
 Penyakit kolagen (Alb. Rendah, Gamma
meningkat)
Contoh Pemeriksaan SPE pada
beberapa penyakit
 Hb Electrophoresis
Normal Hb ;
The protein part of the molecule (globin)
consist of 4 polipeptide chains
Postnatal  at least 3 distinct Hb types
~ Hb A (22)  major normal adult Hb
  - chains &  - chains
~ Hb F (22)  major Hb of the fetus
and the newborn infant
  - chains &  - chains
~ Hb A2 (2,2)  1,5% – 3,5% of normal
adult Hb
  - chains &  -
chains
 Beberapa Metode Mendeteksi
Hb-pati & Talasemia
 Elektroforesis Hb dengan selulose
asetat pH basa.
 Elekroforesis agar sitrat pH asam
 Kuantitasi HbA2.
 Kuantitasi HbF
 HPLC Penukar Ion
Elektroforesis Hb dengan
selulose asetat pH basa
 Skrining
 Buffer alkali (pH 8,2 – 8,6)
 Hb merupakan molekul bermuatan (-)
migrasi ke anoda.
 Hasil yang abnormal dilanjutkan dengan
elektroforesis agar sitrat pH asam.
Elekroforesis agar sitrat pH asam

 Konfirmasi Hb variant.
 Diferensiasi HbS an HbC
 HbC dan S bermuatan (-)
 HbA dan F bermuatan (+)
Kuantitasi HbA2
 Umumnya dilakukan dengan metode
kromatografi penukar ion.
 Densitometer.

Kuantitasi HbF
 Denaturasi alkali (Betke): Akurasi deteksi
HbF hingga 10 – 15%.
 Denaturasi alkali (Singer): 10 – 40%.
 Radial Immunodifusion
 HPLC Penukar Ion
 Sensitif, spesifik, reproducibility lebih
baik.
 Alat: Manual, otomatis.
 Metode didasarkan pada elusi
komponen-komponen Hb berdasarkan
kekuatan ioniknya.
 Pemeriksaan komprehensif.
 Cepat (<10 menit)
THANK YOU
for your attention