Week Four

This week everyone was back in the lab. I continued to do the PCR/gels for genotyping, and I worked on just the beginning steps of another Western Blot for more practice. For Western Blotting, there are two different kinds of staining that can be done to be check that the proteins being sampled are making it through the process properly. Coomassie Blue staining can be done right after the electrophoresis gel separation, and Ponceau S staining can be done after the transfer step that moves the proteins from the gel to the membrane. Since we stopped the process after the separation step this week, we used Coomassie for the staining, whereas last week we used Ponceau since we went through the entire process for Western Blotting. An example of a Ponceau staining can be seen below in a video from Abnova (2010).

Both staining types require the entire gel or membrane to be submerged in the stain, and then destained so only the proteins will stay colored, if they have moved as they should have for the particular step of the procedure. Often the protein ladder is visible and is a marker that the step worked, but it is still good to check that the protein samples have moved since Western Blotting takes so long and the sooner you know to abandon a failed Western Blot, the less time you will waste. However, in most cases Ponceau staining is favored because it destains more efficiently than Coomassie and does not affect the following steps as much, as well as is completed using a membrane which is stronger and less prone to damage than a gel. Either way, staining occurs before antibodies are applied. 

Figure 1. An acrylamide gel stained with Coomassie Blue (Bio-Rad).

The lab technician observed and helped me with my second Western Blot, and it quickly became clear that the lab tech and the graduate student had slightly different methods for Western Blotting. It is incredibly helpful to see different ways to complete tasks so I can develop techniques that works best for me, but it is hard learning different techniques at once when it is a completely new procedure for me. Later, when the lab tech and I asked for a method for a Coomassie staining, we received a few different answers again between the post-doc in our lab and people from other labs on our floor, however, the staining was less exact so we figured out what was best as we went with only a short written procedure the post-doc found as reference.

This week I also helped run a PCR/gel and PCR purification on samples we received so we could then send them to a company for Sanger sequencing. Once the sequencing information was sent to us and we performed a bit of bioinformatics work, we had electropherograms that we were able to look at to find mutations in our samples. My mentor actually found the LMNB1 gene duplication that causes ADLD largely in part from looking at an electropherogram and noticing that it looked strange where LMNB1 is found in samples from patients who had ADLD.

Figure 2. An example electropherogram (Wilson).

Part of the time that the post-doc was gone last week he was in another lab learning a new protocol which he is now trying to encorporate into our lab. Since the protocol is new to us, he needed to order some new reagents, and even though he did not order that many reagents, the order was over $1000. Something I have been learning through my studies and my work in this lab is that scientific research is expensive, which is part of why it is necessary to know what you are doing as a researcher in an attempt to minimize the waste of materials that can cost so much.

References: 

Abnova. 2010. Western Blot - Ponceau S Staining. <https://www.youtube.com/watch?v=Jj_37cDsO7o>.

Bio-Rad. n.d. Protein Stains. <http://www.bio-rad.com/en-us/product/protein-stains>.

Wilson, H. S. n.d. Application of the Polymerase Chain Reaction (PCR) and Bioinformatics in Bacterial Identification. <http://biosci.sierracollege.edu/resources/bio-teaching/pcr-id.html>.