AGAROSE GEL

ELECTROPHORESIS, PAGE
 GEL ELECTROPHORESIS

It is a technique used to separate a mixed

population of macromolecules on the basis of
size, shape and length. It involves running a
current through a gel containing the molecules.
AGAROSE GEL:
 Consists of repeated agarobiose (L- and D-galactose)
subunits.
 Agarose polymers associate non-covalently and form a
network of bundles.
 Molecular sieving is determined by the size of pores
generated by the bundles of agarose in the gel matrix.
 The higher the concentration of agarose, the smaller the
pore size.
 Reagents and Supplies for Gel
Electrophoresis
 Scale - for weighing agarose
 Spatula – for scooping agarose powder
 Flask or beaker - for melting agarose
 Graduated cylinder for measuring buffer
 Microwave or hot plate and large beaker to boil water
 Agarose
 Buffer
 Gel tray(s) and comb(s)
 Gel box(es)
 Power supply
 Photo doc. system
 Reagents and Supplies for Gel
Electrophoresis
Your DNA Sample
Loading dye- for help with loading the gel
DNA Ladder or Marker
20uL Pipettes
Tips
Gloves
Waste container
 BUFFERS

 The most common gel running buffers are TAE (40 mM Tris-acetate,

1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA)

 Add running buffer to the agarose-containing flask.

 EtBr is the most common reagent used to stain DNA in agarose gels

 Alternative stains ---- SYBR Gold, SYBR green, Crystal Violet and

Methyl Blue.
LOADING DYE:

 Loading dyes used in gel electrophoresis serve three major

purposes.

 Add density to the sample, allowing it to sink into the gel.

 Provide color and simplify the loading process.

 Move at standard rates through the gel, allowing for the

estimation of the distance that DNA fragments have migrated.

 What is SDS-PAGE ?

“Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis”

SDS PAGE is a technique for separating proteins based on

their molecular weight. Charged molecule will migrate in
an electric field towards an electrode with opposite sign.
 SODIUM DODECYL SULFATE

 Detergent to give a negative charge to proteins.

 12 carbon hydrophobic end.

 Negatively charged sulfate end.
 POLYACRYLAMIDE GEL

 Composed of polymer polyacrylamides.

 Tight matrix.
 Ideal for protein separation.
 Smaller pore size.
 Proteins much smaller than intact chromosomal DNA.
 Average amino acid = 110 Da
 Protein size is measured in Dalton (Da) or Kilodalton
(kDa).
 MATERIAL REQUIRED

 Protein Samples.
 SDS.
 Buffers.
 Polyacrylamide Gel.
 Staining Solutions.
 Molecular weight markers.
 Electrophoresis chamber.
 Tris buffer to provide appropriate pH.

 SDS (sodium dodecyl sulphate) detergent to dissolve

proteins and give them a negative charge.

 Glycerol to make samples sink into wells.

 Bromophenol Blue dye to visualize samples.

 PROCEDURE
 Incubated sample with SDS at 95C for 4
minutes.
 Denaturation of proteins by heating them with
SDS detergent.
 Membranes will be dissolved.
 Proteins will be unfold.
 Proteins will be covered with negative charges.  

SDS
-----------------------------
-----------------------------
 PROCEDURE
 Reducing agents are used to break the disulfide bonds.
 DTT: Di thio threitol
 OR
 BME: Beta mercapto ethanol
PROCEDURE
 PROCEDURE

 Negatively charged protein samples are loaded

into wells at the top of the gel.

 An electric current is applied so that there is a

positive charge at the bottom of the gel and a
negative charge at the top of the gel.
PROCEDURE
 PROCEDURE
 The negatively charged
s-s
proteins will migrate to SDS, heat
the bottom of the gel
proteins with
according to size. SDS

 Larger proteins migrate

slowly. -
 Smaller proteins migrate
quickly through the gel.
 Proteins separate by
molecular weight and +
size.
 STAINING

 Place the gel in the staining solution for 30

minutes.
Coomassie Blue or Silver Stain

 Stain the gel until the bands are properly seen.

 Determine the approximate molecular weight of

the visualized protein bands.