In immunochemistry, as in baking, repetition and attention to varying outcomes leads to perfection of the technique.
Contributed by Crystal Stutzke
Although reproducibility is currently a huge topic in science and research, problems with reproducibility affect many aspects of our lives.
Recently I had a conversation with my mother-in-law about the difficulty replicating her chocolate chip cookie recipe and it made me think of the reproducibility crisis in the scientific community. My mother-in-law makes the best chocolate chip cookies ever, and like many other people who have killer cookies, she shares her recipe with others. The only unfortunate issue with recreating this delectable treat is I can’t reproduce that taste or look of grandma’s amazing cookie. I mean how hard can it be… they are cookies!! To really kill my ego further my mother-in-law can come in my kitchen and reproduce her cookies to the exact awesomeness as she makes at her place. It left me wondering what am I doing wrong? She was quick to make two very important points: one… it was not her recipe, and two… It wasn’t perfect at the beginning. That is when the large metaphorically blinking “REPRODUCIBILITY” neon sign was above my mother-in-law’s head.
Performing immunoassays, like baking chocolate chip cookies, requires repetition and finesse to achieve consistent, high quality results.
There are so many variables in making cookies: the ingredients used, how they are mixed together, the temperature and time they are baked are just a few key factors in perfecting the chocolate chip cookie. Well the same can be said for protein applications like IHC and Western Blotting. To have success in reproducing the same awesome result each and every time one should practice and repeat the process many times to have an understanding of each and every step of the process.
Documenting and observing errors in your procedure can provide invaluable data for future experiments.
Let’s use Western blots for an example. Here are the major steps: 1) Lysate preparation 2) SDS-PAGE 3) Protein Transfer 4) Antibody Incubation 5) Development. In each of those major components there are several teeny tiny (yet critical) steps one must optimize and understand for the entire process to be a success over and over again. Have you ever realized you’ve made a mistake (and could identify it) mid-way through an experiment, document it in your lab notebook and continued on with the experiment, taking note of the end result? Well if not, you should!!! That is an important part of the learning process, also known as a form of optimization. You shouldn’t necessarily just toss an experiment because you made a mistake. Sometimes it is a good idea to see what happens when you didn’t use the proper lysing agent in your lysis buffer or when you left your lysates at room temperature for an extended period of time. What happens when you accidentally add DMSO instead of BME to your sample buffer? Have you ever used transfer buffer instead of running buffer for SDS-PAGE? How about when you forget to add Tween 20 in the TBST for washing and incubating? Or when you use mouse secondary antibody to detect for a rabbit polyclonal antibody?
Mistakes build knowledge, troubleshooting prowess, and reproducibility.
When you have achieved successful repetition of different protein applications over and over it builds knowledge and confidence. So when you change variables and don’t have success it allows to you quickly identify the issue and moves you successfully forward in your research.
Further Reading
Are Monoclonal Antibodies Really More Specific?
1% SDS is the Lysis Buffer of Choice for Most Western Blots