Well the ‘big experiment’ today turned out to be the ‘big anti-climax’… In work at 8 am to start preparing for the experiment – using a technique called Isothermal Titration Calorimetry (ITC) to look at the change in heat caused by the binding of a protein to another structure or ligand.
I was supposed to start the experiment at 9.30, but when concentrating my proteins, the inside of the concentrating tube for the centrifuge shattered, meaning i had to pick out pieces of plastic so i could recover my protein solution – not a good start. After last night’s issues regarding the proteins (see yesterday’s post), i also had to run a protein gel (SDS-PAGE) to confirm the proteins were in fact present. In between centrifuge runs, i had some simple solutions to prepare too. The second issue arose when trying to quantify how much protein was in my concentrated samples. The spectrophotometer we use just wasn’t giving us a sensible reading, it was suggesting we had more protein than we started with. At this point it was 10.30 already, so we went down to the ITC to begin the experiments, despite not knowing the protein concentration.
The ITC takes a lot of preparation before a sample can be loaded, with lots of intricate parts that need cleaning with multiple solutions. It is a complicated machine so i will try to significantly condense the info on what it does:
- Has a reference cell, with water.
- Has a sample cell, containing your protein of interest.
- Has a remote controlled syringe, which does controlled and staged injections of the ligand for your protein.
- The machine measures the heat change when binding between your protein and its ligand occurs, against the water reference.
Essentially, the ITC can be used to produce a lot of data on protein ligand binding, and is a crucial technique in drug discovery and many other fields!
After preparing the ITC, and setting up the first experiment, it was 1 pm. I went for lunch for an hour, then returned to set up the second experiment. We return to find that the result shows little binding activity. We decide to run a control experiment to try and determine why this is. Whilst this is running, i go and type up my own protocol for the ITC, then me and my supervisor check the protein gel from this morning. This unfortunately suggests that the protein used for the ITC was 1/16 of the concentration it needed to be, probably explaining the poor result.
It was 5.30, so i went to finish up the experiment, clean all of the equipment and bring my equipment back up to the microbiology lab. After doing this and chatting to a couple of the guys down in NMR for a little bit, it is now 6.45. I leave work by 7!
Something really refreshing my supervisor has mentioned to me in the last few days is that he thinks my time management is bad, and that i could be more efficient, and that i should be more efficient so i can spend more time at home! It is something i am really going to work on, and one of the reasons i started #PhDaily, to make myself more accountable for the time that i spend at work. Although today was long, and very tiring, it is nice to end today’s #PhDaily on a positive note!
Finally (just remembered), also a weird feeling today, as my supervisors have decided to split my PhD in half, and are applying for a new PhD student to work on one of two proteins that i was working on. Kind of sad, i had become attached to it! But the amount of work for me to do over the next few years was just piling up!
Thanks for reading