Question

What is the purpose of each of the four steps outlined in the Background section for today's lab?

Background: This week you will get data that you can analyze to address your working hypothesis. This procedure involves several steps. First you will soak your blots in a solution containing milk proteins and the "primary antibody" which is specific for green fluoresent protein (anti-GFP). Next, you will add a "secondary antibody" (goat anti-mouse HRP) which can bind to the primary antibody AND is itself attached to an enzyme will use to catalyze a reaction, generating a chemiluminescent product that you can detect with X-ray film.

Answer 1

The Background section in the lab is discussing the "Western Blotting" technique.

The Western Blotting technique is a procedure done to identify or track down a specific protein from a mixture of proteins that were isolated from tissues or cells. This technique involves two main procedures. They are: (1) Separation of proteins from cells or tissues and (2) Identification of proteins.

Separation of proteins is done by gel electrophoresis.

Identification of protein is done by Blotting technique also called Immunoblotting technique.

PURPOSE OF EACH OF THE FOUR STEPS DISCUSSED IN THE LAB:

STEP 1: Soaking blots in a solution containing milk protein:

REASON: This step 1 is called "Membrane Blocking". This procedure is done to block the unbound or free spaces in the membrane (that is, the space that is not bounded by sample protein - the protein that has to be identified), so that the primary antibodies will not be able to bind in these free spaces. The problem of primary antibody binding in the free spaces is that it will produce high background signal that may interfere with the interpretation.

This membrane blocking is done after transferring of the sample protein on to the membrane. Once the sample protein is added to the membrane, it will bind at specific places in the membrane. Later, the unbound or free spaces in the membrane are made to be bound with non - specific milk protein.

STEP 2: Soaking the membrane with primary antibody

REASON: The primary antibody added will recognize and bind to the specific sample protein.

STEP 3 : Soaking the membrane with primary antibody specific for green fluorescent protein.

REASON:

The reason for adding the primary antibody specific for green fluorescent protein is for obtaining chemiluminescence. The chemiluminescence method is widely used due to its high sensitivity. Chemiluminescence method is a quantitative method, meaning, it helps to determine the accurate amount of protein in the sample. However, today, fluroscence based Western Blotting technique is considered to be more quantitative. Also, chemiluminescence allows for multiple film exposures.

STEP 4: Addition of secondary antibody

REASON: After washing the membrane to remove any unbound primary antibody, secondary antibody that is specific for the primary antibody is added. Normally, the secondary antibody is conjugated with an enzyme, especially HRP (Horse Radish Peroxidase). This is done in the presence of a substrate that is luminol based. This enzyme will react with the luminol substrate which is a sensitive substrate and emits light that allows visualization using X-ray film.