The SDS-PAGE

Dr Suresh
 What is SDS-PAGE?

SDS-PAGE
(= sodium dodecylsulphate-
polyacrylamide gel
electrophoresis)

-method for separation of

proteins according to their
molecular weight
SDS PAGE is a technique for separating proteins based
on their molecular weight.

Large proteins migrate slowly through the gel matrix and

small proteins migrate quickly through the matrix

The porous gel matrix is composed

of the polymer polyacrylamide
 Link

polymerization Cross Link

The polymerization reaction is catalyzed by

TEMED (tetramethyl Ethylenediamine and
APS (ammonium persulfate)
 PAGE

 Gels are cast by polymerizing a solution of acrylamide

monomers into polyacrylamide chains
 Gel pore size can be varied by adjusting the concentrations
of polyacrylamide
 Smaller proteins migrate faster than larger proteins through
the gel
Native proteins
SDS (sodium dodecyl sulfate) binds to and coat the
protein
The hydrophobic region of SDS binds to the
hydrophobic region of proteins and unfolds them.

This also covers the proteins with a negative charge.

All proteins now have a net negative charge.

-----------------------------
SDS
-----------------------------
 SDS

 all polypeptide chains are then forced into extended

conformations
 SDS treatment eliminates the effect of differences in shape
 individual polypeptide chains migrate as a negatively
charged SDS-protein complex through the porous
polyacrylamide gel
 speed of migration is proportional to the size of the proteins
 smaller polypeptides running faster than larger polypeptides
How about covalent link?

DTT/Me SH

S-S

HS
 Disulfide bonds must be broken to
completely unfold the protein.
Reducing agents/antioxidents are used to break the disulfide bonds.
DTT: Dithiothreitol
and
BME: Beta mercaptoethanol - - - -
- -
S S -
-
SDS -
S ------------------- S -
----------------------- - -

S -----------------------------
SDS
S -----------------------------
Plus DTT
 Protein loading buffer
 Protein gel loading buffer contains Tris buffer to maintain
constant pH
 glycerol to increase sample density,
 the strong ionic detergent SDS (sodium dodecylsulfate),
 β-mercaptoethanol, a reducing agent. . Beta-mercaptoethanol
eliminates disulfide bonds in proteins by reducing them (adding
hydrogen atoms).
 Heating
-proteins denatured by heating them
in a sample loading buffer containing
sodium dodecyl sulphate (SDS)

-the proteins no longer have any

secondary, tertiary or quaternary
structure
-resultant proteins take on a rod-like
shape and a uniform negative charge-
to-mass ratio proportional to their
molecular weights
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.

The negatively charged proteins will migrate

to the bottom of the gel according to size.
 Stacking gel
 To obtain optimal resolution of proteins, a “stacking” gel is
poured over the top of the “resolving” gel.
 The stacking gel
lower concentration of acrylamide (larger pore size),
lower pH
different ionic content
 This allows the proteins in a lane to be concentrated into a
tight band before entering the running or resolving gel
 produces a gel with tighter or better separated protein bands
Visualizing the proteins:
Types of Stains:
1. Coomassie Blue – Sensitivity of 0.1ug protein per band

Traditional method requires staining followed by destaining to remove

background gel staining.
GelCode Blue from Pierce does NOT stain the gel and requires no
destaining. Increased sensitivity of 25ng protein per band

2. Silver Stain – Sensitivity of 2ng protein per band

Glassware and plasticware must be very clean to prevent stain from

precipitating out of solution.

Prone to high background due to impurities in the acrylamide and

surface artifacts such as fingerprints
Lane 1. Kaleidoscope Markers
2. Shark
3. Salmon
4. Trout
5. Catfish
6. Sturgeon
7. Actin and Myosin Standard
 Comparison of the sensitivity of
SDS PAGE stains:
Identical SDS-polyacrylamide gels
were stained with

A) SYPRO Orange protein gel stain

B) SYPRO Red protein gel stain
C) silver stain
D) Coomassie brilliant blue stain
Outline of the experiment
*Prepare polyacrylamide gels
*Add diluted samples to the sample buffer
*Heat to 95C for 4 minutes
*Load the samples onto polyacrylamide gel
*Run 200 volts for 30-40 minutes
*Stain in Coomassie Blue stain
*Destain
*Identify molecular markers, proteins