Blotting Techniques made easy

Description

Part 1:

Definition and introduction

3 types of Blotting techniques

   Southern

   Northern

   Western

Sothern: 6 steps

    DNA Digestion

    Gel Electrophoresis

   Blotting

   Probelabelling

   Hybridization

   Detection

Sothern blotting Diagram

Part 2:

Sothern Blotting explanation

Part 3:

Sothern Blotting 10 steps

Factors affecting

Stringent conditions

Membrane for bloat transfer

Applications

Part 4:

Applications

strengths and limitations

Part 5:

Northern Blotting

steps: 8

RNA isolation

probe generation

Agarosegelelectrophoresis

immobilization

Hybridization

washing

detection

re-probing

diagram

Part 6:

Norther blotting applications

advantages and disadvantages

Part 7:

Western blotting

Dentition

protocol

steps

Western blotting buffer preparation:

sample preparations

Gelelectrophoresis

protein transfer

Membrane blocking

antibody incubation

western blotting detection

Data analysis

W.B diagram

Part 8:

procedure

explanation

Part 9:

what is SNOW Drop

Detecting Phosphorylation States of Proteins

Many 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 Groups

Detecting 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 Points

Western 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 Proteins

Many 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 Proteins

Some 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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