All Because of a Harmless Western Blot

Ben Ferguson -- Sometimes, the simplest things can test your patience and your sanity. At times, you wonder how you ever were able to do them in the first place.

While in Los Angeles a few weeks ago visiting a collaborator’s laboratory, I was in a car accident during the morning rush hour. (I’m totally fine -- just a scrape on my forehead -- but my glasses unfortunately suffered fatal injuries and have been reincarnated as a pair of hip, exorbitantly-priced specs.) For the next week, I was kind of terrified to drive. I drove ten miles under the speed limit, stopped for a three-count at all stop signs, white-knuckled 9 and 3 o’clock, and let people go in front of me in heavy traffic. You know, things everybody does on a regular basis, including me. I felt as though I was 16 again and just learning how to drive. The world moved so fast! How could I ever have driven with such little attention paid to the road and yet been in so few accidents?

In the lab, I’ve done roughly the same experiment six times over the past four days: a Western blot -- one of the most ubiquitous, simplest experiments in research and one that virtually everybody working in basic science knows how to do. Well, I’m 0-for-6. It was a week full of ... profanities?

I’ve done plenty of Western blots in my research career. When I was a summer researcher early during medical school, I did them regularly and successfully. But I cannot do it now, and it’s driving me nuts. The problem with these sorts of things is that they can be so simple, or perhaps that we can get so accustomed to doing relatively complex things, that we do them without thinking. They often have many steps to them too, each with their own chance of failure. By the last step, the compounded chance of failure is enormous. When the experiments fail, we must take a step back and examine why they -- or we -- failed.

Did I make the gels correctly? Are my units right? Is the glassware clean enough? Which cables am I supposed to use again? Are the protein estimations accurate? How old are these antibodies? Did I not transfer long enough? What’s with that funky noise coming from the developer? Did I touch the membrane too much? Wait, do I even know how to operate this pipette properly, or have I been doing this wrong all along?! WHAT IS A PIPETTE ANYWAY?? It’s enough to drive a person mad, especially when you ask these questions over and over, make what you think are the appropriate changes, and still fail. To make matters worse, some of these things are within your control; some are clearly not, at least not immediately.

This leads to a cycle of overthinking things. Overthinking the most minute of details that may, but probably don’t, have anything to do with the outcome of the experiment. Overthinking which combination of changes you need to make in order to do it right the next time. Overthinking how terribly long all of these repeats are taking you, and overthinking how you might speed up the process (which is almost never a good thing to do in the lab in times of distress). Overthinking precisely how and why you’re overthinking these things and overthinking how you can stop overthinking about overthinking these things.

It’s a damn mess. All because of a harmless Western blot.