SDS PAGE

&
WESTERN BLOTTING
By,
KRISHNAKUMAR M S
I-M.Sc., Biochemistry
Government Arts College-Paramakudi
PAGE ELECTROPHORESIS
 PAGE - Poly Acrylamide Gel Electrophoresis
 A technique used to separate protein based on size

and charge using electric current

 Ulrich K laemmli discover the PAGE in 1970.
 PAGE is used in,
 molecular biology studies
 peptide mapping and protein quantification
 Forensic studies
 To perform western blotting
TYPES OF PAGE
The PAGE is used in two ways,

 1. Native PAGE
Based on Size and Charge

 2. SDS – PAGE - Sodium Dodecyl-Sulfate Poly

Acrylamide Gel Electrophoresis
Based on Size
PRINCIPLE
 The protein is –ve charged due to SDS and moves towards +ve
charge.
 Smaller protein moves faster and farther to bottom of the gel
 Larger protein moves slower
 The proteins are separated based on their size.
COMPONENTS:
 APS: Ammonium Per Sulphate
2 types of gel
-Initiates polymerization of gel
-Stacking gel-Higher Porosity(Ph-
 TEMED: Tetramethyl
6.8),5% acrylamide
ethylenediamine
-Separating gel-Lower Porosity(Ph-
-Catalyze polymerization of the gel
8.8),12% acrylamide
 Buffer
 Acrylamide and Bis acrylamide
-Tris HCL and counterions (glycine) to
-Polymerize to form the gel
stabilize the process and electrophoretic
-Carcinogenic and Neurotoxin
mobility
 Sample protein Bromophenol blue – Tracking dye
 SDS

-An Anionic Detergent

-Denatures protein structure
-Coats the protein –ve charge
 β mercaptoethanol

-Breaks the disulphate bond (S=S)

PROCEDURE
 Prepare sample(Protein+SDS+β
mercaptoethanol+Glycerol+Bromophenol blue)
 Set the glass slides with spacers between(vertical)
 Prepare the separating gel and stacking gel
 Place the comb to create well
 Fill the electrophoresis tank with buffer
 Load the sample into the wells
 Apply electric current and run the gel
 When the dye reaches the bottom stop the current
 Take the gel stain it for visualization
 Staining cosmasic brilliant blue, silver staining
APPLICATIONS
 It is used to measure the molecular weight of the molecules.
 It is used to estimate the size of the protein.
 Used in peptide mapping
 It is used to compare the polypeptide composition of
different structures.
 It is used to estimate the purity of the proteins.
 It is used in Western Blotting
 It is used in HIV test to separate the HIV proteins.
 Analyzing the size and number of polypeptide subunits.
 To analyze post-translational modifications.
BLOTTING TECHNIQUES:
 The process of transfer of DNA or RNA -PVDF-Poly vinylidene Fluoride

or Proteins from gel to membrane is Membrane

called blotting.  Blocking agents(BSA) are added for

OVERVIEW: to avoid non specific binding

 Samples are separated by gel  Hybridization/Antibody: Probes are

electrophoresis added to specific to sample

 The samples are transfer to a membrane  Visualization: Autoradiography and

-Nitrocellulose membrane and Nylon Chemiluminescence

membrane
TYPES OF BLOTTING :
 There are basically 4 types of blotting:

 Southern blotting.(for DNA)

 Western blotting.(for Protein)
 Northern blotting.(for RNA)
 Eastern blotting.(for PMT Proteins)
WESTERN BLOTTING:
 The detection of specific protein using blotting
technique
 W. Neal Burnette discover the western blotting
 Also called as Immuno blotting technique
 Use antibodies as probe
 The protein separate in SDS PAGE
 Sample Transfer to membrane(Electro blotting)
 Antibody binding to specific protein
 Protein detection
ELECTRO BLOTTING
 Electric voltage is applied  The buffer helps to
transfer of protein transfer the protein
 The proteins move

towards the +ve charge

 The proteins are

Imprinted on the
Nitrocellulose membrane
 The foam pad and paper

helps to hold the gel and

membrane together
BLOCKING AND ANTIBODY BINDING
 The blot is incubated with Dry or,
milk or BSA to block other  With a enzyme such as
sticky places of membrane HRP(Horseradish Peroxides)
 Primary antibody(ab-1) specific
to protein target is added
 Unbound antibodies are washed
away
 Secondary antibodies (ab-2) are
added specific to the Fc region
of Primary antibody
 The secondary antibodies
contain the radio labeled isotope
PROTEIN DETECTION:
 The radio labeled probes attached are detected using
Autoradiography
 other modes of detection are,
 Chemiluminescence: In which a enzyme is attached to

the probe upon treatment with substrate, color changes

are light emitted.
APPLICATION
 Confirmative test for HIV and HBV
 Definitive test for Bovine Spongiform Encephalopathy
(Mad Cow Disease) and Lyme disease
 Diagnosis of various metabolic disease
 Estimation of the size of the protein as well as the
amount of protein present in the mixture.
LIMITATION:
 Very delicate and time consuming process (min 6 hrs)
 Incorrect labeling of protein can happen due to the
reaction of secondary antibody
 Well trained technicians are required for this technique
EASTERN BLOTTING:
Detection of Post Translational Modified Proteins
Ishikawa & Taki discover the eastern blotting

Same as western blotting

Same SDS PAGE

Same electro blotting technique

Same blocking procedure

Differ in Antibody binding technique

-Antibody specific to PTM

-which target the site of PTM like Methylation, Acetylation, Lipids and
etc.
Same detection
APPLICATION:
The technique has been used to identify and purify different plant products.
Eastern blotting also allows the detection of modifications in proteins of

different origins.
It also helps to study the nature of interactions between different molecules

by the use of ligands.

Eastern blotting has been extensively used to compare modifications in

proteins obtained from different bacterial species.

It is also used to detect carbohydrate epitopes in different proteins.