Protein Techniques

Goals:

Outline SDS-PAGE, Western blot and Immunofluorescence experiments and apply them to relevant examples 

Apply the above mentioned techniques to experimental questions (and know why to use each one)

Probe Hybridization

A probe is a complementary to the DNA/RNA of sequence. This probe is a labeled complementary strand of DNA or RNA, and can specifically detect a sequence of interest in a complex mixture.

Credit: MacGraw-Hill Animations (https://www.youtube.com/channel/UCxUHVv2k31uTOiCm4njuRfQ)

Antibodies

Antibodies are a protein complex that can recognize a specific region called an epitope on a given protein which is referred to as the antigen.

Antibodies are generated by injecting an animal with a protein of interest (POI), the animals own immune system then produces antibodies that are able to recognize the foreign protein of interest, and then researchers collect a blood sample, and purifies the antigen-specific polyclonal antibodies.

These antibodies can be used in techniques as the antibodies with the POI detects the protein, and a secondary antibody which is labeled (fluorescence) binds to the first antibody (known as the primary antibody), and allows for visualization of the location of the protein of interest.

Credit: ThermoFisher Scientific (https://www.thermofisher.com/ca/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/introduction-secondary-antibodies.html)

SDS-PAGE

SDS-PAGE is a techinique which detects the expression of a gene at the protein level. SDS-PAGE stands for SDS-PolyAcrylamide Gel Electrophoresis.

This technique is done by adding heat, SDS, and Dithiothreitol (DTT) which ultimately denatures the protein, coats it in a uniform negative charge, and breaks the disulfide bonds of the protein. The protein is visualized using a non-specific dye such as: Coomassie, Silver Staining, Ponceau.

Credit: https://www.youtube.com/channel/UCHy7KdzL2qEFWB4hJtjXb7w

Western Blotting

Western Blotting uses the SDS-PAGE technique from above to run the proteins out on a gel, then the proteins (just as is done to RNA using Southern Blotting) is transferred to a membrane which immobilizes the proteins, and the protein is then incubated with a primary antibody, then if necessary is incubated with a secondary antibody (if indirect detection is required) to detect the proteins via fluorescence.

Credit: https://www.youtube.com/channel/UC_S2ilNrvdhmGcaatLtejGg

Immunofluorescence

Is a technique which allows to visualize a specific protein in cells. The cells are incubated with a specific primary antibody, and the incubated with a secondary antibody, and then is detected using fluorescence microscopy.

Credit: https://www.youtube.com/channel/UC_S2ilNrvdhmGcaatLtejGg

Comparison of the Techniques

SDS-PAGE: Is used to visualize differences in expression of abundant proteins.

Western Blotting: Gives the sizes of a specific protein, and allows for quantitative analysis

Immunofluorescence: Gives a proteins position in a cell, and is not quantitative.