Western Blots from Scratch

The last few weeks have been a whirlwind here in the molecular neuroscience lab. Every step of the project has been meticulously planned out from ground zero. Where we lack in experience, we have made up for with lots of reading, troubleshooting, and persistence. Once we decided on a treatment plan for the mice, injections were performed and tissue was harvested. Since there are so many different protocol variations based on which protein one is interested in, we had to be very careful when deciding how to prepare the tissue samples for western blotting. After experimenting with several different lysis methods, we decided on one which we thought would be best and processed all of our hippocampus (HPC) samples. From there, we had to learn how to protein assay. This learning curve was much more mellow compared to the other steps of the process, and we were able to quantify the protein concentrations of all our HPC samples on one plate after running a practice plate. Now we could start our westerns!

The western blot is a two-day process with a large number of steps, and a mistake at any one of those steps can completely ruin your results. We have run several practice blots with some small successes, but we have a lot to improve on and will continue to try and get our experiment optimized. Once we have our protocol with concentrations and times dialed in, we will be able to start accumulating some serious data!

This project has definitely shown me how unified the Department of Biological Sciences is here at ISU. Since we lack experience with this fairly complex experiment, we have reached out to and been helped by many outstanding individuals here in the biology building. The members of the Rose lab —Kole and Alex— have been very willing to share protocols and advice on preparing gels and running westerns. The members of the MCRF upstairs —especially Lisa— have also been extremely gracious and have provided assistance when learning how to run protein assays and imaging our gels. Additionally, the stockroom, the Groome lab, and the Pilarski lab have all helped us get our hands on the equipment that we need to be successful. I can’t thank these bengal scientists enough for all of the help!"

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