(Solution) SCIE1046: BIOS242 Activity 6.3: Western Blot Transfer: Prepare for protein detection

COURSE  

SCIE1046: Fundamental of Microbiology with Lab

About the Lab

Learning Objectives:

  • Build a transfer stack with the correct orientation.
  • Set up a transfer tank to transfer protein from an SDS-PAGE gel to a membrane.
  • Critique the quality of protein transfer using a removable protein-identifying stain.

Estimated Length: 35 to 45 minutes

 

MAKE THE CONNECTION

The background information in section 2 was adapted from the following Microbiology lecture course Tutorials:
5.4.1 Antibody Production and Laboratory Testing
5.4.2 In Vitro Assays

 

2. Background Information

The following background information will be helpful as you prepare for the simulation.

2a. Immunoblot Assay

Protein gel electrophoresis can be followed by the addition of antibodies to identify specific proteins. This technique, described in the steps below, is called the Western blot.

 

STEP BY STEP

Step 1. Polyacrylamide gel electrophoresis (PAGE), is used to separate proteins on an electrophoresis gel.
Step 2. Protein antigens in the gel are transferred to a nitrocellulose membrane, immobilizing them.
Step 3. The nitrocellulose membrane is exposed to a primary antibody produced to bind to the protein of interest.
Step 4. A second antibody equipped with a molecular beacon (i.e., something that makes it visible) is added to bind to the first antibody (primary antibodies can also be tagged with a molecular beacon in some cases). Molecular beacons can be enzymes that will react with a chromogenic substrate to produce a color if the antibody is present. They can also be fluorophores (molecules that fluoresce when excited by light). The beacon indicates the location of the specific protein in the membrane.

The image below shows the steps of a Western blot in part (a) and an example of a Western blot test for antibodies to HIV in part (b). The bands on the Western blot indicate the presence of the protein of interest, which takes up stain.

Polyclonal antibodies are generally used for Western blot assays because they are able to bind to multiple epitopes of the primary antigen and are therefore more sensitive than monoclonal antibodies. However, monoclonal antibodies can also be used when there is a need to target specific epitopes.

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