ELECTROPHORESIS

DNA
PROTEINS

A technique where charged molecules migrate under the

influence of electric field
How charged molecules separate in medium under electric field?
 Ions move in electric field

 Velocity α Charge on ion

 Velocity α Voltage applied

 Matrix is porous

 Pores retard molecules on basis

of their size

 Velocity is inversely dependent on

size of molecule
 Before you start: Impart a charge on DNA or protein sample

How would you impart a charge on proteins or DNA in sample?

Use of buffer of pH 8.6 all

molecules are –ve ly charged
Types: On the basis of electrophoretic apparatus:

 Horizontal electrophoresis

 Vertical electrophoresis

 Tube gel electrophoresis

Types:On the basis of electrophoretic medium:

 Agarose gel electrophoresis

 Polyacrylamide electrophoresis

 Cellulose acetate electrophoresis

 Isoelectric focussing
Horizontal Agarose Gel Electrophoresis for
proteins
 APPARATUS
 Horizontal electrophoretic apparatus

 E/P apparatus consist of :

 EP tank
 filled with buffer
 EP plate
 migration of charged species takes place
 Two electrodes
 -ve ly charged molecule migrate to +ve
electrode
 At pH 8.6 all molecules are –ve ly
charged
 so they migrate to the +ve electrode
 What is Agarose?
•Linear polysaccharide

•Repeat units of agarbiose

•Gal & Anhydrogalactose

•Dry Powder

How is gel prepared

from agarose?
•Mix Buffer, Boil, Cool

•Pour
on E/P Plate with
Comb

•Cross Links  Pores formed

•More agarose  More C/L

•1% agarose is used

 Digging the wells for pouring the sample

Where do we load the sample on gel?

Will sample not dissolve?

 Make sample heavy

 GLYCEROL

 Indicator dye (amido black)

 Well no. 1 = N sample + dye

 Well no. 2,3,4 = sample to analyze + dye

Apply the voltage

 Smaller proteins will migrate

faster

 After 20-45 minutes dye on

margin

 Current is switched off.

 Gel is put in stain

 (Amido black or coomassie)

 Gel is destained using acetone.

 Gel is destained but not proteins

 Proteins will emerge as stained

bands.
Which serum proteins are clinically useful,
when analyzed by electrophoresis ?
Serum electrophoresis
 Major components
 Prealbumin,
 Albumin
 α1 antitrypsin (AAT)
 α2 macroglobulin (AMG)
 Haptoglobin
 β lipoprotein (LDL)
 Transferrin
 Complement
 Fibrinogen (in plasma)

 Minor components
 Ceruloplasmin
 Hemopexin
 α1 acid glycoprotein
 C-reactive protein.

 Major and minor proteins form 6 bands on Serum is used not Plasma
serum electrophoresis. Serum proteins are synthesized in liver
Except Ig and protein hormones
Proteins in 6 bands
Densitometric scan of electrophoretic gel
 Serum protein abnormality profile 1

Hypoalbuminemia due to starvation/malnutrition

 ↓ in proteins synthesized by liver

 ↓ in Ig secreted by lymphocytes

 General ↓ in intensity of ALL bands

 MOST PRONOUNCED IS
Hypoalbuminemia (< 2 g%)
 Serum protein abnormality profile 2

Hypoproteinemia due to protein losing enteropathy (PLE)

 Hypoproteinemia in absence of
 proteinuria (kidney disease) or
 defect of protein synthesis (liver
disease)

 It is due to
 Mucosal ulcer (peptic ulcer)
 Mucosal damage (celiac disease)
 Lymphatic obstruction

 All bands diminished

 Except α2 band
 Because AMG, haptoglobin are APR
Acute phase reactants
 ↑ in stress or inflammation
 Infection, surgery, trauma, necrosis

 APR are
 α1 antitrypsin (AAT)
 Complement
 α1 acid glycoprotein (AAG)
 CRP
 Haptoglobin
 Fibrinogen

Why are they increased?

 AAT  counter proteolytic enzymes
 Complement  part of immune response
 AAG  part of immune response
 CRP  scavenger
 Haptoglobin  binds Hb during hemolysis
 Fibrinogen  seal gaps in wall integrity
 Serum protein abnormality profile 3

Nephrotic syndrome
 Loss of LMW proteins in urine
 Example is Minimal Change disease

 MCD
 Loss of foot processes

 LMW Proteins lost in urine are:

 Albumin, AAT, Transferrin, IgG

 Proteins retained are:

 AMG and micelle of LDL

 Serum finding:
 ↓ Albumin, α1, β1, γ band
 ↑ α2, β2

Why β2 is increased? NS is c/b Hypoproteinemia and Hyperlipidemia (LDL)

 Serum protein abnormality profile 4

Cirrhosis of liver

 Hepatocellular damage

 ↓ albumin synthesis
 ↓ oncotic pressure

 Compensatory response is to ↑
polyclonal synthesis of Ig
 Oncotic pressure slightly increase

 γ band enhanced
 Serum protein abnormality profile 7

Monoclonal gammopathy (Multiple Myeloma)

 Monoclonal proliferation of plasma

cells

 ↑ Ig (Ab) in serum

 M band on serum electrophoresis

(paraproteins)

 Bence Jones proteins on urine

electrophoresis (Bence Jones proteinuria)
 Agarose Gel E/P
for DNA
1% agarose & buffer pH 8.6

Sample + dye + glycerol

1st well  DNA marker

2,3,4, etc.  DNA sample

Apply current

Small DNA fast & vice versa

Stain with Ethidium bromide 

Fluoresce

Put Gel on UV Transilluminator

Estimate size of DNA fragment

Cellulose acetate E/P

 Cellulose acetate strips.

 Separation on the basis of charge.

 Substitute for gel when there are

large number of samples

 Used for confirmation of Sickle

Cell Anemia
Vertical PAGE
 Polyacrylamide gel electrophoresis

 Plate is held vertical in tank.

 Used mainly for separation of proteins.

 It has got high resolution.

 (separate proteins differing in even one
amino acid.)

 The gel used is polyacrylamide.

 Like agarose they form cross links &

very fine pores.
Vertical PAGE.
 Also called SDS-PAGE.
 Tank, plates, combs & electrodes.

 Space between the plates

 gel is poured.
 Sample treated with
 Mercaptoethanol (denature 30)
 SDS (denature 20)
 Protein molecule becomes linear

 Two SDS molecule can combine with one

amino acid
 swamps the original charge
 mass to charge ratio of all proteins is equal.

 This property makes the separation of

proteins on the basis of mass alone.
Vertical PAGE
 Make wells using combs.

 Well no 1
  Protein marker (known MW)

 Well no 2,3,4
 sample

 Electric current is applied

 Proteins separate
 on the basis of their mass.

 Stain the gel with Coomassie

 Then destain it.

 Bands of proteins are now visible.

THANK YOU