SDS Page , 2D Page and Iso-electric Focusing

1. SUBMITTED BY
Vivek kumar
M.sc MICROBIOLOGY
Bangalore university

2. SDS PAGE
 It is most common method for separation of proteins
based on their molecular masses.
 This is a form of denaturing electrophoresis.
 The sample molecule are first denatured and then
subjected to electrophoresis.
 The detergent SDS(Sodium dodecyl sulphate) is used.
 It consist of hydrophobic chain and polar sulphated head.

3.  The hydrophobic chain can intercalate in to
hydrophobic interior of the protein and disrupt its
compact structure.
 SDS coats protein with a uniform negative charge and
causes them to migrate to the anode.
The net intrinsic charge of the protein is cancelled.
 The protein move with a velocity inversely
proportional to their molecular mass.
 SDS page is done in discontinuous gel systems
consisting of two gels:
STACKING GEL and RESOLVING GEL

5. STACKING GEL
 It is of pH 6.9.
 Its purpose is to concentrate the sample components
into this bands.
 The gel has a low percentage of acrylamide (3-5%)
,with low ionic strength.

6. RESOLVING GEL
 It has high percentage acrylamide (8-20%) with higher
ionic strength and alkaline pH.
 This gel achieves separation of molecule as they move
through it. Usually Tris-Glycine buffer is used.

8. ADVANTAGES
 Widely used due to its simplicity and versatility.
 Molecular mass of protein can be obtained.

9. DISADVANTAGES
 Two proteins having same molecular mass can not be
separated.
 If protein deviate from ideal globular shape or if they are
bound above or below average amount of SDS, inaccurate
mass estimate will result.
 So it is necessary to use standard proteins to compare the
molecular mass.
 Post translational modification also affect mobility of
proteins.

11. ISO ELECTRIC FOCUSING
 Net charge of protein is highly influenced by the
pH.
 At low pH, most proteins are positively charged.
 At high pH, they are negatively charged.
 The pH value at which proteins have no net charge
is the isoelectric point(PI).

12.  A stable pH is established in a solution using a
mixture of ampholytes.
 Ampholytes are synthetic low molecular mass
polymers having varying PI.
 When such a mixture is placed in an electric field
they form a pH gradient.
 When a protein is mixed with pH gradient and
subjected to electric field, it will migrate towards
the PI.

13.  At pH above PI it will have net positive charge and
below PI it will have negative charge.
 The net charge is 0 and no electric mobility.
 This experiment is called IEF as it involves focusing of
molecules at their individual PI values.
 IEF is usually conducted in glass tubes or in horizontal
gels. Precast IEF gels are available.
 PI may be determined by running a mixture of known
protein of known PI value.

15. 2-D PAGE
 Two proteins of same molecular mass can be
separated.
 This is a combination of IEF and PAGE.
 Proteins are separated in first dimension by IEF.
 This is performed in 9M urea in rod shaped gels.

16.  Then the IEF gel is placed along the tops of SDS
gel and electrophoresis is done.
 Proteins separated in IEF is based on their PI value.
 2nd dimension mainly on the basis of molecular
mass.
 2-D PAGE is based on the assumption that it is
unlikely that 2 proteins will have same PI and
molecular mass.