Southern, Northern and *Western Blotting · 2014-01-08 · Southern Blotting •A technique which...

Southern, Northern and *Western Blotting

Gaye Güler Tezel, MD,PhD Hacettepe University School of Medicine

Department of Pathology

Basic Molecular Pathology Course with “Hands On” Interactive Practice September 19-21, 2013, Istanbul

Plan

• Introduction

• Principle

• Protocol

Southern Blotting

• A technique which allows the detection of a specific DNA sequence in a large, complex sample of DNA (e.g. cellular DNA).

• Named after the British biologist Edwin M. Southern in 1975.

• Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization.

Principle

• The Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples.

• The Northern blot is a technique used in molecular biology research to study gene expression by detection of RNA.

• The Western blot (alternatively, immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

• Southern blot- DNA

• Northern blot- RNA

• Western blot- Protein

• Western blot analysis can detect one protein in a mixture of any number of proteins.

Western Blotting-I

• It was introduced by Towbin in 1979.

• It is also called immunoblotting, uses antibodies to identify spesific protein targets bound to a membrane.

Western Blotting-II

• Proteins are separated using SDS-polyacrylamide gel electrophoresis which separates proteins by size.

• Immobilize protein onto a permenant substrate (Nitrocellulose/PVDF membrane).

• Identify protein of interest.

Western Blotting-III

• Seperation of proteins- gel electrophoresis

• Transfer to the membrane

• Blocking the non-spesific binding sites

• Antibodies

• Development-visualisation

Protocol

• A high-quality antibody directed against a desired protein is used as a probe to detect the protein of interest.

Step I

• Seperation of proteins

*The first step in WB procedure is to seperate the proteins in sample by using denaturing gel electrophoresis (SDS-PAGE)

PAGE

Step 2

• Transfer to the membrane- blotting

• The actual blotting process may be active (electroblotting) or passive (capillary).

• Electroblotter is used for faster and more

efficient transfer of protein from gel to membrane.

• The proteins are transferred to a sheet of special matrix called membrane (nitrocellulose/PVDF)

• Sandwich of filter paper, gel, membrane and more filter paper is prepared in a cassette, which is placed between electrodes.

• An electric current is passed through the gel causing the proteins to electrophorese out of the gel and onto the membrane.

Nitrocellulose membrane

• Nitrocellulose membranes are the most popular matrix used in protein blotting.

• Most proteins can be successfully blotted using a 0.45 μm pore size membrane.

• For proteins of low molecular weight or peptides, a 0.2 μm pore size membrane is recommended

PVDF Membrane

• PVDF membranes are used for immobilization of proteins, due to its non-specific affinity for amino acids.

• It has 0.22 or 0.45 micrometres pore sizes

Filter paper

• Filter Papers are pre-cut cotton sheets for wet or semi-dry, passive or electrophoretic transfer of proteins from polyacrylamide gels (SDS-PAGE) to PVDF, nitrocellulose, or other membranes.

• Sheets of blotting paper are necessary components of transfer sandwiches and cassettes that typically must be assembled for various kinds of protein or nucleic acid, gel-to-membrane, transfer protocols.

Transfer Buffers

• Common buffers used for Western blotting are;

– Towbin buffer (25 mM Tris-HCL pH 8.3, 192 mM glycine, 20% methanol)

– CAPS buffer (10mM CAPS pH10.5, 10% methanol)

Blotting Equipments

Buffer tank

Cooling unit

Electrode assembly

Gel holder casette

Buffer tank lid

Sandwich Model

foam pad

filter paper

membrane

gel

filter paper

foam pad

• The proteins retain the same pattern of separation they had on the gel.

Transfer Methods

• There are four major ways to transfer macromolecules from gels to membranes: – Diffusion blotting

– Vacumm (capillary) blotting

– Wet (tank) blotting

– Semi-dry blotting

Step3-Blocking the membrane • Cellular proteins will bind to the

membranes.

• Antibodies are also proteins, and they can also bind non-specifically to the membranes.

• To control this undesirable binding, membranes have to be “blocked” using a generic protein (such as BSA, skim milk) which binds to any remaining sticky places on the membrane.

Step4- Antibodies • After blokage of non-spesific binding

sites, add primary antibody which binds your protein of interest.

• The secondary antibody will be directed toward IgG of the species in which the primary antibody was raised. In almost all cases, the secondary antibody will be conjugated to horseradish peroxidase (HRP).

Step5-Visualisation

• HRP is an enzyme that catalyzes the conversion of an “enhanced chemiluminescent” substrate into byproducts and light.

• ECL substrates allow for the detection of extremely minute quantities of protein.

Western Blot

• Remove gel from the gel-cast and place into transfer buffer.

• Cut filter paper (larger than your gel)

• Cut a piece of PVDF membrane, soak in methanol for a 5 seconds, and rinse it in transfer buffer.

• Place three pieces transfer-buffer soaked filter paper on the blotting apparatus.

• Place the membrane onto the paper (no air bubble)

• Place the gel on top of the membrane in the correct orientation. The marker should be on the same side that it was when you loaded the gel.

• Gently roll out any air bubbles.

• Place three more pieces of filter paper on top of the gel.

• Set the transfer apparatus to 80V for 2-3 hours or 8V for overnight.

Sandwich Model

foam pad

filter paper

membrane

gel

filter paper

foam pad

• Incubate the membrane with primary antibody.

• Wash the membranes with TPBS.

• Incubate the membrane with secondary antibody.

• Wash the membranes with TPBS.

• Incubate the membranes with ECL solution for 1 minute.

• Wrap the membrane and expose to X-ray films.

Southern Blot

Western Blotting

• Do not touch the membrane without gloves.

• Do not let the membrane dry out.

• Roll out any air bubbles during transfer step.

• Be careful to electrode position.

Thank you ..