SDS PAGE
&
WESTERN BLOTTING
By,
KRISHNAKUMAR M S
[Link]., 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.