Juan Manuel Gonzalez Morena: Controls are more important than a beautiful blot

Juan studies how tumor suppressors kill cancer cells, works as a postdoc at IOCB Prague and as Head of Biology at Taveren Therapeutics, and runs a Western blot almost every day. We talked about the work, about experiments that don’t work and tips which could be useful to all scientist.

What do you do, and what does a normal day in the lab look like for you?

J: I am a cancer biologist. Most of my work is about understanding the molecular mechanisms that tumor suppressors use to kill cancer cells. I am also involved in looking for new compounds that increase the expression of these tumor suppressors.

My work is a mix of experiments, experimental design and project coordination, so every day is a bit different. Normally I start with cell culture and treatments, then I prepare samples and run Western blots or other biochemical assays. Part of the day I spend analysing data and deciding what the next experiment should be.

Has that changed over time?

J: Yes. As my role became more senior, I spend more time discussing results with colleagues, troubleshooting experiments and coordinating collaborations.

What are you working on right now?

J: Mainly cancer biology. I’m interested in tumor suppression and how changes in the levels of specific proteins affect cancer cell survival. I’m also working on projects with small molecules, where we try to understand their mechanism of action.

So protein analysis is a big part of your work.

J: It is. Western blot is the technique I use almost every day. I use it to look at changes in protein expression, protein stability and signalling pathways involved in tumor suppression.

I also work a lot with LACTB, which is a mitochondrial tumour suppressor. Western blotting has been important in these projects, both to monitor its expression and to study the biological changes that come after it.

Do you remember an experiment that didn’t go to plan?

J: Definitely. I was trying to detect a low-abundance endogenous protein. I expected a reasonably clear band, because that’s what I always saw in the papers I read for that project. But the signal I got was very weak or inconsistent.

How did you find out what was wrong?

J: I usually split the problem into levels. First I ask if it could be biological. Is the protein actually expressed in the conditions of my experiment? Then I look at sample preparation, so concentration, degradation, lysis buffer. After that I check if my usual transfer method is suitable for that protein. And at the end I look at the antibody and detection: antibody concentration, the buffer for the antibody, blocking buffer and exposure.

In the end it was partly biological and partly technical. In my conditions the protein was not much expressed, and the antibody gave a very low signal, even for the positive controls.

How did you find out what was wrong?

J: I usually split the problem into levels. First I ask if it could be biological. Is the protein actually expressed in the conditions of my experiment? Then I look at sample preparation, so concentration, degradation, lysis buffer. After that I check if my usual transfer method is suitable for that protein. And at the end I look at the antibody and detection: antibody concentration, the buffer for the antibody, blocking buffer and exposure.

In the end it was partly biological and partly technical. In my conditions the protein was not much expressed, and the antibody gave a very low signal, even for the positive controls.

Who or what do you turn to when you’re stuck?

J: Usually the people around me first. A colleague who already worked with the same protein or the same antibody can sometimes solve very quickly something that would take me a few more experiments to understand.

To find methods and small experimental details I use PubMed, Google Scholar and ResearchGate. For very practical troubleshooting I sometimes use Reddit too. There are scientific communities where people talk about real problems they had at the bench, and you can find useful tips there that you will probably never find in a paper.

But in the end there is always some trial and error. The important thing is that it’s controlled trial and error. Change one parameter at a time and write down what you changed.

What would you tell someone who is just starting with Western blot?

J: Controls are more important than a beautiful blot. Always have a positive control if you can. Otherwise, when something goes wrong, you never know if the problem was the sample, the transfer, the antibody or the detection.

And don’t overexpose the blots just because the bands look nicer. A saturated band often tells you less than a weaker signal that is still in the range where you can measure biological changes.

You tried VerdiBlot in your lab. What difference did you notice compared with your previous membrane?

J: Mainly the signal-to-background ratio. When I compared VerdiBlot side by side with the PVDF we normally use, the background was much lower and the bands were sharper.

It also worked well when we stripped and re-probed it. That’s important for us, because we usually look at several proteins from the same sample.

Did anything surprise you?

J: A few things. First, how easily it fitted into our protocol. I expected that I would need to change or optimise some steps, like transfer conditions or blocking buffers. But we just used it in our normal workflow.

Another small but practical thing is that VerdiBlot doesn’t need to be activated in methanol like PVDF. It’s a small step, but it’s one less source of variation.

And one thing I really didn’t expect. VerdiBlot was compatible with Blue Native transfer, and the Coomassie dye didn’t stay on the membrane much, while on PVDF it was much more visible. That made the protein detection much cleaner.

You work both at IOCB and at a startup. What's the biggest difference between the two, or is it actually quite similar? Was there anything that surprised you? If so, can you tell us more about it?

J: There are actually quite a lot of similarities, because in both cases the work is driven by scientific results. The main difference I can see so far is how the priorities are defined. In the startup, there is a very clear translational goal, so the experiments and resources have to be prioritized with that final objective in mind. Since the company is still at an early stage, we are also setting up many of the workflows and helping to shape how the project develops. What I really like is being able to see the project from a broader perspective and understand how each part of the company contributes to the final goal of eventually developing something that could reach the clinic and help patients.