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Edit Comment Close Premium member Presentation Transcript Blotting techniques : Blotting techniques Presented by Birendra kumar contents : contents Introduction Steps involve Gel electrophoresis Blotting Technique Types Northern Blotting Southern Blotting Western Blotting Conclusion Steps involved : Steps involved There are five main steps involved:- Sample preparation Gel electrophoresis Blotting Hybridization Dectection Sample preparation : Sample preparation Gel electrophoresis : Gel electrophoresis Movement of charged particle in an electric field. Used in separation or isolation of bimolecule Gel may be of SDS, agar, agarose. There are two main buffer used- TAE and TE buffer Blotting techniques : Blotting techniques Transfer of DNA, RNA or Proteins, typically from a electrophoresis gel to a membrane e.g. nitrocellulose. This membrane can then be subject to further techniques such as hybridization HYBRIDIZATION: Process where two complementary single strands of nucleic acid (DNA or RNA) form a double helix. Using specific probes that are labelled specific sequences of DNA can be identified. Types of blotting : Types of blotting DOT BLOTTING: hybridization of cloned DNA without an electrophoretic separation is called DOT BLOTTING CAPPILARY BLOTTING: hybridization of cloned DNA with an electrophoretic separation and transfer of it to membrane through capillary action of alkaline. Types of blotting : Types of blotting There are three main hybridization techniques which vary in the sample blotted and the probes used: Northern Blot-Transfer of an RNA sample separated and identified using DNA or RNA probes. Southern Blot-Transfer of an DNA sample separated and identified using DNA or RNA probes. Western Blot- Transfer of an Protein sample separated and identified typically using an antibody. Southern blotting : Southern blotting a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels, however non-radioactive alternatives are now available. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines. Northern blotting : Northern blotting to study gene expression by detection of RNA (or isolated mRNA) in a sample. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues. Western blotting : Western blotting analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodiesspecific to the target protein. Steps in nothern blotting : Steps in nothern blotting 1.1 Tissue preparation 1.2 Gel electrophoresis 1.3 Transfer 1.4 Blocking-Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) inTris-Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. 1.5 Detection- During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour Detection : Detection applications : applications The main use of this technique is to identity any changes in DNA sequencing or genes expressed, e.g. comparing genes expressed by a diseased cell to genes expressed by an healthy cell. Other uses include- Testing for hereditary disease, Evolutionary history of species, Screening e.g.food supply Applications to synthetic biology - identification of various parts in natural organisms, -?more? Application in bioinformatics : Application in bioinformatics Thanks you : Thanks you You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.