BLOTTING

TECHNIQUES

What is blotting?

Technique for transferring • DNA • RNA • Proteins

onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.

TYPES OF BLOTTING TECHNIQUES

BLOTTING TECHNIQUES

Southern Blot

It is used to detect the DNA.

Northern blot

It is used to detect the RNA.

Western blot

It is used to detect proteins.

Blotting sheet• Whatman 3mm paper….. world’s most widely used

blotting paper.

REASON????• high quality• purity • consistency

(0.34 mm) is used extensively in electrophoresis for lifting of sequencing gels.

Creating the Sandwich

• The sandwich consists of :filter paper 

Nitrocellulose membrane

gel matrix

another piece of filter paper

SOUTHERN BLOTTING

History:• Named after Sir Edwin

Southern

• Developed in 1975

SOUTHERN BLOTTING

“Used to detect the DNA”• This method Involves:SeparationTransferHybridization.• This DNA can be:• Single gene• Part of a larger piece of DNA……..viral genome

“The key to this method is Hybridization”

Hybridization

“Process of forming a dsDNA molecule between a ssDNA probe and a ss-target

patient DNA”

PRINCIPLEThe mixture of molecules is separated. Immobilized on a matrix. Probe addition to the matrix to bind to the molecules. Unbound probes are removed.

“The place where the probe is connected corresponds to the location of the immobilized

target molecule.”

Steps in southern blottingThe DNA is digested

Fragments

Gel electrophoresis

Transfer to membrane

Probing

Autoradiogram

APPLICATIONS

Southern blotting is used in:• Gene discovery • Mapping• Evolution • Development studies• Diagnostics • Forensics

Northern BlottingHistory:

Northern blotting was developed by James Alwine and George Stark at Stanford University.

Northern blotting is a technique for detection of specific RNA sequences

Steps involved in N.BRNA isolation

Loading of sample on Agarose gel

Blotting on nitrocellulose membrane

Labeling with probe

Washing to remove unbound probe

Detection by autoradiogram

APPLICATIONS• A standard for the direct study of gene expression at

the level of mRNA (mRNA transcripts)• Detection of mRNA transcript size• Study RNA degradation• Study RNA splicing - can detect alternatively spliced

transcripts• Study RNA half-life• Study IRES (internal ribosomal entry site) – to

remove possibility of RNA digestion vs. 2nd cistron translation.

Western blottingDiscovery???• Dr. Douglas Lake of the University of Arizona School of

Medicine's Department of Microbiology and Immunology

“A technique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody.”

Principle

This technique works on the principle on

“Antigen-Antibody” relationship

Prerequisite for W.B

The SDS PAGE technique is a prerequisite for Western blotting.

“SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it.”

Steps in W.B1. Gel electrophoresis:

The proteins are separated according to size.

2. Membrane Transfer: Transferring to nitrocellulose by applying current.

3. Blocking: Done to prevent non-specific protein interactions between the membrane and the antibody protein.

Blocking in W.B• The blot is incubated with a generic protein (such as

milk proteins) which binds to any remaining sticky places on the nitrocellulose.

• An antibody that is specific for the protein of interest(the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen.

•  Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules .

Cont..• Following several rinses for removal of non-

specifically bound Ab1, the Ab1-antigen complex on the nitrocellulose sheet is incubated with a second antibody (Ab2), which recognizes the primary antibody and binds it.

• Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-Ab1-Ab2 complex

Applications• To identify the specific proteins

• To identify their masses

• The confirmatory HIV test to detect anti-HIV antibody in a human serum sample.

• The definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').

• Some forms of Lyme disease testing also employ Western

blotting. 

Protein gel (SDS-PAGE) that has been stained with Coomassie Blue.

Faster Way to Western Blot

• The Quick Western Kit provides a universal detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody incubation step.

• This kit works with a variety of primary antibodies.

• This reduces the overall time to complete a Western blot.

Standard Western Blot