Blotting Blotting The term Blotting refers to the

Blotting

Blotting • The term “Blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. • Techniques for transferring DNA, RNA and proteins onto a carrier so they can be seperated, and often follows the use of gel electrophoresis.

Types of blotting Southern blotting Northern blotting Western blotting

Principle • Edward M. Southern (1975) • Identification of particular size of DNA from the mixture of other similar molecules • Principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization • Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis. Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using specific DNA probe that is complementary to the desired DNA

Procedure/ Steps • Restriction digest: by RE enzyme and amplification by PCR • Gel electrophoresis: SDS gel electrophoresis • Denaturation: Treating with HCl and Na. OH • Blotting • Baking and Blocking with casein in BSA • Hybridization using labelled probes • Visualization by autoradiogram

Application of Southern blotting • Southern blotting technique is used to detect DNA in given sample. • DNA finger printing is an example of southern blotting • Used for paternity testing, criminal identification, victim identification • To isolate and identify desire gene of interest. • Used in restriction fragment length polymorphism • To identify mutation or gene rearrangement in the sequence of DNA • Used in diagnosis of disease caused by genetic defects • Used to identify infectious agents

Northern Blot • RNA blot • Study gene expression • Developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University

Procedure

Application • Observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment

Western blot • Protein immunoblot • PAGE

Procedure • SDS-PAGE • Membranes (nitrocellulose, nylon, and polyvinyldifluoride (PVDF)) • Passive (or capillary) transfer and electroblotting. • Passive – sandwich of filter paper soaked with transfer buffer, gel, membrane, and more filter paper, 1 -2 days • Electroblotting- sandwich of filter paper, gel, membrane, and more filter paper is prepared in a cassette, which is placed between platinum electrodes, 1 -4 h

Detection of Blotted Proteins

Advantages • Need for only small reagent volumes, • Short processing times, • Relatively inexpensive equipment • Ease of performance

Results