Dr. Shadi Shams’s lab.

Last week I had the opportunity to visit the labs of Dr. Tatiana Baldari. It was very inspiring to see lab techniques that I had learned about in freshman biology be used to work on scientific breakthroughs. The main techniques that we saw were the Western Blot followed by immunoblotting, which is the technique used to label the proteins that have been separated on the gel and membrane. Since Shadi has covered the process of Western Blotting and transferring the data onto a membrane, I’m going to look at the process which tags and identifies the proteins that are present.

Once a gel has been run and all the proteins have separated, the researcher needs to identify which proteins are actually present in that particular sample. The molecule used to tag the proteins is called an antibody. Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest. Normally, this is part of an immune response, but in this case they are harvested and used as sensitive and specific detection tools. After blocking, a process that that prevents the antibodies from binding to the membrane directly, a dilute solution of the primary antibody is incubated with the membrane. The solution is composed of buffered saline solution with a small amount of detergent. This is sealed together over-night.

The following step is to use a secondary antibody to bind and tag the primary antibody so it can be detected. The membrane is first rinsed to remove any unbound primary antibody and it is then exposed to a different antibody which is directed at a species-specific portion of the primary antibody. Antibodies tend to come from animal sources, for example an anti-rat secondary will bind to just about any rat primary antibody. This allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and provides far more consistent results. The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.

This secondary antibody is then detected using multiple methods. The most popular are radioactive and fluorescent detections. Other methods include a colormetric and chemiluminescent detection.

It was very interesting to visit Dr. Baldari’s (or should I say Dr. Shams’ due to her deep knowledge of how the lab worked) lab and see the different projects that they are working on. The main focus of the lab was the P66 Shc gene and different parts of the lab were working on different aspects of the gene. One was looking at how it affects cells in leukemia patients while another part looked at how they affected mast cells. I look forward to the chance of doing performing some of these techniques for myself tomorrow.