Troubleshooting and Bacterial Proteins

Throughout my time working with bacterial protein, I learned the importance of troubleshooting- that is figuring out why your experiment may have yielded strange results. As I reviewed the methods for creating a western blot, I came up with four main reasons why it may not have turned out the first time: the proteins may have failed to transfer to the membrane, the substrate may not have been functioning, the film may not have developed, or our antibodies may not have interacted with the proteins correctly.

We first performed a Coomassie Stain stain to make sure our proteins transferred to the membrane. After letting it sit for a few minutes, we saw blue bands spanning across the membrane. Eliminating the possibility that our protein may not have transferred from the gel to the membrane, we then tested the substrate that interacts with the horseradish peroxidase enzyme on the secondary antibody. When exposing the substrate to another enzyme we saw that it glowed brightly, proving that our substrate was functioning properly. Finally we used a different developer and fixer when transferring our proteins onto the film, and we saw that the film still remained empty. After narrowing down these possibilities we concluded that it was the antibody that was not functioning properly.

This past week, I made another western blot, this time using a new primary and secondary antibody, and on part of the membrane I added a loading control antibody, which binds to RNA polymerase, a protein that should be consistent across all of the strains of bacteria. After successfully moving the bacterial proteins onto the membrane and adding the antibodies, it was time to develop our films. In the dark room, I exposed the proteins and antibody to the substrate and in less than a minute, several bands were already glowing brightly. With these promising initial results, I finished developing the film. However, upon closer examination I noticed that the lane with the E. coli strain was the only lane where we could see the black bands. Even the loading control films, which should have had bands consistent across all samples, lacked evidence of the proteins in the pseudomonas strains. After discussing these results with my mentor and a post doc in the lab we came to the conclusion that the E. coli was not only producing more LasR, a protein receptor in quorum sensing, than the pseudomonas, but it was also producing more of RNA polymerase. These strange results can be linked to the plasmid that was inserted in our strain of E. coli to force the over production of LasR. The mechanism by which it does this also increases the production of RNA polymerase, leading to our skewed results in the loading control.

From this experiment, I not only learn more about quorum sensing and the LasR receptor, I also learned of the almost infinite factors that have the potential to affect an experiment. Through troubleshooting, I was able to effectively narrow down these many possibilities and create a successful western blot, however I also have discovered new factors that have an effect. In the future I would want to adjust the concentration of our antibodies as well as try a new loading control antibody that does not bind to RNA polymerase.