Anyone who follows me on Instagram may have noticed that last week I uploaded a picture of a western blot. Now, trying to get a western blot to work has been a bit of a saga for me, my lab mates and family are very aware of how frustrating it has been. It's a technique that I had never done before and is not routinely done by anyone in my lab, so it has been a bit of a trial and error process. I’ve spoken to people, attended an online course, read protocols, and so on but I was never getting a good result. I purchased a new batch of my primary antibody, played around with incubation times, different extraction methods, and different membranes, but still nothing. Recently, I decided to give the process a go using a different antibody, not the one most relevant to my research, but just to check to see if I could get something. And then…low and behold... I got a nice looking blot with clean, sharp bands in all the right places! It really was a cracking feeling and provided a real confidence boost as it implied that some of the issues were with the antibody and not my technique. I then tried another blot using the original antibody and, whilst not particularly clean and sharp, (although this is likely due to reusing a stored primary antibody solution), I got bands in the correct places for the first time. Now, this didn’t make too much sense, I am sure that I did not do anything noticeably differently and to be honest, was expecting no bands and would then contact the supplier to discuss a potential issue with that antibody batch.
I discussed this with my supervisor, sharing the good news that with a different antibody, my westerns were working, and how I now, rather peculiarly, had potential progress with the original one. I explained that my protocol was unchanged and that I was unsure why I would suddenly start seeing results. He responded by telling me a story from his PhD, where a certain extraction always seemed to come with the dame contaminant, regardless of how many times he performed it, or attempted to tweak the process. But then one day, at a different institute, performing the exact same protocol, the contaminant was gone. He told me that science just isn’t always an exact science. Sometimes things just start to work. Either something completely outside of my control has changed, resulting in the western finally working, or (and preferably) I am doing something different/better. Either way, it is difficult to know which of these is the case but, for the time being, I will ride the high of this small win.
I have heard similar tales from people within my lab who have done the same qPCR over and over again without it working properly and then all of a sudden, boom, it works. Others have told me of similar experiences with western blots, cloning, PCRs, and every other lab technique you can think of. So, I guess my supervisor is right, apparently, science is just not an exact science. Sometimes things don’t work and then with no clear rhyme or reason, they do. It is certainly frustrating and can be downright disheartening. But when it finally clicks and the infra-red scanner gradually reveals those sharp, clean bands in your western blot, it is incredibly satisfying. I guess there are probably life lessons to be gleaned from this experience surrounding perseverance, self-belief, or something along those lines. But right now, I am enjoying the motivation and confidence boost that comes with this small win. This short post is just to give an insight into PhD life and give an example of how frustrating research can be but also how exciting it is when something you’ve been stuck on, finally works.
I hope you have enjoyed reading this little story of my western blot saga, as always, I welcome any and all feedback and would love to hear any similar stories people have from their own research. Please do leave a comment or ping me an email.
I discussed this with my supervisor, sharing the good news that with a different antibody, my westerns were working, and how I now, rather peculiarly, had potential progress with the original one. I explained that my protocol was unchanged and that I was unsure why I would suddenly start seeing results. He responded by telling me a story from his PhD, where a certain extraction always seemed to come with the dame contaminant, regardless of how many times he performed it, or attempted to tweak the process. But then one day, at a different institute, performing the exact same protocol, the contaminant was gone. He told me that science just isn’t always an exact science. Sometimes things just start to work. Either something completely outside of my control has changed, resulting in the western finally working, or (and preferably) I am doing something different/better. Either way, it is difficult to know which of these is the case but, for the time being, I will ride the high of this small win.
I have heard similar tales from people within my lab who have done the same qPCR over and over again without it working properly and then all of a sudden, boom, it works. Others have told me of similar experiences with western blots, cloning, PCRs, and every other lab technique you can think of. So, I guess my supervisor is right, apparently, science is just not an exact science. Sometimes things don’t work and then with no clear rhyme or reason, they do. It is certainly frustrating and can be downright disheartening. But when it finally clicks and the infra-red scanner gradually reveals those sharp, clean bands in your western blot, it is incredibly satisfying. I guess there are probably life lessons to be gleaned from this experience surrounding perseverance, self-belief, or something along those lines. But right now, I am enjoying the motivation and confidence boost that comes with this small win. This short post is just to give an insight into PhD life and give an example of how frustrating research can be but also how exciting it is when something you’ve been stuck on, finally works.
I hope you have enjoyed reading this little story of my western blot saga, as always, I welcome any and all feedback and would love to hear any similar stories people have from their own research. Please do leave a comment or ping me an email.
