Essentials of Anatomy & Physiology, 4th Edition Martini / Bartholomew PowerPoint ® Lecture Outlines prepared by Alan Magid, Duke University The Muscular System The Muscular System 7 7 Precented to: Dr Rashid Presented by: M. Zeeshan Arid No. 17 arid 4330 Blotting Techniques

CONTENTS Blotting Def. Types of Blotting Southern CONTENTS Blotting Def. Types of Blotting Southern Blotting Method Applications Northern Blotting Method Application Western Blotting Method Application

What is blotting?  Blots are techniques to separate DNA, RNA and proteins so they can be separated, and often follows the use of a gel electrophoresis.

TYPES OF BLOTTING TECHNIQUES Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein.

SOUTHERN BLOTTING Professor Sir Edwin Souther Biochemistry Professor Sir Edwin Souther Biochemistry Won Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences. Won Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences. Professor Sir Edwin Southern

Cont…. This method Involves separation, transfer and hybridization. This method Involves separation, transfer and hybridization. It is a method routinely used in molecular biology. It is a method routinely used in molecular biology.

Cont…. Southern blotting combines agarose gel electrophoresis with probe hybridization. Southern blotting combines agarose gel electrophoresis with probe hybridization. The key to this method is Hybridization. The key to this method is Hybridization. Hybridization - Process of forming a double- stranded DNA molecule between a single- stranded DNA probe and a single-stranded target patient DNA. Hybridization - Process of forming a double- stranded DNA molecule between a single- stranded DNA probe and a single-stranded target patient DNA.

APPARATUS

Whatman 3MM paper nitrocellulosemembrane

Capillary plotting apparatus

Steps in southern blotting 1. Digestion 2. Gel electrophoresis.

Cont…. 3. Restriction fragments denatured with alkali. 4. DNA fragment are single stranded. 5. Blotting(nylon membrane)

Cont….6.Hybridization The probe hybridizes to the complementary DNA restriction fragment. The probe hybridizes to the complementary DNA restriction fragment.

Cont…. robe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized. 7, Probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized.

APPLICATIONS Southern blots are used in gene discovery, mapping, evolution and development studies, diagnostics and forensics ( It is used for DNA fingerprinting, preparation of RFLP maps) Southern blots are used in gene discovery, mapping, evolution and development studies, diagnostics and forensics ( It is used for DNA fingerprinting, preparation of RFLP maps) identification of the transferred genes in transgenic individuals, etc. identification of the transferred genes in transgenic individuals, etc. Southern blot is used to detect the presence of a particular bit of DNA in a sample Southern blot is used to detect the presence of a particular bit of DNA in a sample

Northern Blotting Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting

Steps involved in Northern blotting 1. Isolation (various tissues, various developmental stages of same tissue etc.)

Cont…… 2. Agarose Gel run The resulting gel following after the electrophoresis run. The resulting gel following after the electrophoresis run.

Cont…… 3. Blotting ( Amino Benzyloxymethyl filter paper)

Cont…… 4. Hybridization. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. 5. The membrane is washed to remove unbound probe.

Cont…… 6. The labeled probe is detected via autoradiography, this results in the formation of a dark band on an X-ray film. Now the expression patterns of the sequence of interest in the different samples can be compared. Now the expression patterns of the sequence of interest in the different samples can be compared.

APPLICATIONS A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts) A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts) Detection of mRNA transcript size Detection of mRNA transcript size Study RNA degradation. Study RNA degradation.

Western Blotting  Introduced by Towbin et al in  Western blotting is a method of detecting a specific protein from a protein mixture by the use of labeled antibody.antibody  western blot is also known as an immunoblot.

Steps 1.Proteins are extracted from the sample. 2.Polyacrylamide gel electrophoresis. 3.Separated molecules are transferred into a PVDF membrane or nitrocellulose membrane by electroporation

The membrane is blocked for nonspecific binding with the antibodies. Transferred proteins are bound with primary antibody (enzyme labeled antibodies). The membrane is washed to remove nonspecifically bound primary antibodies. Bound antibodies are detected by adding a substrate and detecting the coloured precipitate formed.

Application  It is useful in the detection of anti- HIV antibodies in a human serum sample.  It can also be used as a confirmatory test for Hepatitis B infection and definitive test for mad cow disease.

SouthernNorthernWestern On name of inventor Misnomer Separation of DNA RNAProtein Denaturation needed No needNo Need Nitrocellulose filter membrane Amino benzoyloxymethyl filter paper Polymer Sheet DNA-DNA hybridization RNA-DNA hybridization Protein to Antibody capillary transfer. electric transfer. Differences