Friday, June 22, 2012

Week 5-Western Blots Galore!

My primary focus this week was continuing the development of various Western Blots. One of the members of our research team, Abeer, came back this week from maternity leave to continue her project. The lab was especially busy this week because a lot of buffer solutions had to be prepared in order to practice the cardiomyocyte isolations that will begin next week.

One thing I really enjoy about this research experience is learning (and practicing) various biological lab techniques that was only briefly mentioned in my biology class. Before entering the lab, I had only heard of a "Southern blot." This technique is used to detect a specific DNA sequence in DNA samples and uses probe hybridization. Even though the names are similar, a Western blot is completely different. It targets the identification of specific proteins in a given sample through gel electrophoresis and antibody hybridization.

The first step in a Western blot is to prepare the tissue using a sonicator (there are various techniques in preparing the tissue, but the primary one used in the lab is with a sonicator). The sonicator breaks down the solid cell wall of the cells in the particular sample and enables the protein found within the cell to be freed.

The next step is to perform gel electrophoresis. This separates the proteins of the sample by electric charge and molecular weight. Smaller proteins move faster through the gel and the proteins are separated by size. The proteins move through a specific agar gel; the more concentrated the agar gel is, the better the resolution of the lower molecular weight proteins. (agar gel is created using a powder, diluent, and heat - similar to jello!) Conversely, the lower the concentration, the better the resolution of higher molecular weight proteins. The samples are loaded into walls in the gel, with one lane reserved for a marker. This marker is a commercially made mixture of proteins having pre-determined molecular weights. They are also stained to produce highly visible and colored bands.

Running Buffer
Samples to be Loaded

Loading Samples

Loading Samples

Electrophoresis with an Electric Current

After running the gel, the proteins are transferred to what is called a membrane (PVDF paper). The primary method for transferring the proteins to the membrane is a process known as electroblotting.  This uses an electric current to to pull proteins from the gel into the PVDF membrane. What I find really unique about this process is that the electric current pulls the proteins from the gel onto the membrane in the exact same order and size as on the gel!  As a result of this process, the proteins are exposed on a surface and this is used for detection.

Next, steps must be taken to prevent the antibody from binding to a non-target protein. This technique is called blocking and it involves using TBST (Tris-Buffered Saline with Tween 20). The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. When the antibody is added, there is no room for it to attach other than on the binding sites of the specific target protein.

Detection may be perhaps the most important step in the entire process and is when antibody hybridization occurs.  In my lab, we practice this in two steps: a primary and secondary antibody. The PVDF membrane is probed with a particular antibody (mouse antibody, rabbit antibody, donkey antibody, etc.). When a match occurs, a different color on the membrane is produced.

Last, the membrane is incubated with ECL (a solution used in chemiluminescence) which allows for chemiluminescent detection. The light is detected by a photographic film and results are printed.

Other activities this week included autoclaving glass bottles to fill them with buffers and practicing cardiomyocyte isolations in order to adequately perform next week's activities.

Autoclave Unit

Autoclaved Bottles 
Cardiomyocyte Isolation


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