Overview

Part 1:Definition and introduction3 types of Blotting techniques Southern Northern WesternSothern: 6 steps DNA Digestion Gel Electrophoresis Blotting Probelabelling Hybridization Detection Sothern blotting DiagramPart 2:Sothern Blotting explanationPart 3:Sothern Blotting 10 steps Factors affectingStringent conditionsMembrane for bloat transferApplicationsPart 4:Applicationsstrengths and limitationsPart 5:Northern Blottingsteps: 8RNA isolationprobe generationAgarosegelelectrophoresisimmobilizationHybridizationwashingdetectionre-probingdiagramPart 6:Norther blotting applicationsadvantages and disadvantagesPart 7:Western blottingDentitionprotocolstepsWestern blotting buffer preparation:sample preparationsGelelectrophoresisprotein transferMembrane blockingantibody incubationwestern blotting detectionData analysisW.B diagram Part 8:procedureexplanationPart 9:what is SNOW DropDetecting Phosphorylation States of ProteinsMany proteins are post-translationally modified by kinases, which add phosphate groups to their substrates. Western blotting is great for detecting the presence of phosphorylated proteins. Phosphorylated proteins become heavier, due to the added weight of the phosphate group, so they often migrate more slowly than their un-phosphorylated forms.Detecting Changes in Protein Levels Across Treatment GroupsDetecting the presence and absence of a protein may seem like a simplistic assay, but when each sample represents a different treatment group, western blotting can be very informative about the amount of each protein in treatment. This also applies to detecting how a certain treatment changes the post-translational modification of a protein (i.e. phosphorylation, ubiquitination, etc.).Detecting Changes in Protein Levels Across Time PointsWestern blotting is incredibly informative for determining the effect of time on a protein. For example, if each sample is a protein mixture of cells that are in different phases of the cell cycle, then western blotting will reveal how much a protein is present or absent during each phase.Detecting Truncated Isoforms of ProteinsMany proteins are cleaved in order to be activated, or have naturally occurring truncation isoforms. Each isoform may have a different level of activity, a different target protein, or represent a different cellular state. Western blotting is great for detecting the ratio of truncated to normal isoforms of a protein.Detecting Tagged ProteinsSome proteins are engineered, through the process of molecular cloning, to contain short sequences of amino acids that serve as a tag. Common tags include the HA-tag and the Myc-tag. These tags serve as a foreign protein epitope that does not naturally occur in the biological system being studied. Thus, the tag makes the protein easy to detect compared to all other naturally occurring proteins. An antibody directed to the tag will identify the presence and amount of the tagged protein in the western blot.and many more in slide explanation

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