Dundee Research Interest Group

Another busy two weeks in the lab have flown by and it is quite unbelievable that I have been working at MRC PPU for 2 months now.

In my first few weeks, most of the protocols I have been carrying out have already been tried and tested by many scientists before which has meant I have been getting lots of satisfaction from obtaining clear, good quality results from experiments that have worked first time. This has been a great boost to my confidence but perhaps not a true reflection of life as a research scientist. Many of my colleagues will have to work for weeks, even months, systematically optimising their experimental protocols and eliminating sources of error or doubt in order to obtain results that are of publication quality. Behind each figure (a figure is anything outside of the main text of the paper that one may refer to aid understanding, this includes diagrams, images, data, equations etc) in every scientific paper lies hundreds of hours of bench work and head scratching and relies on the ability of scientists to problem solve and maintain optimism even when an experiment may not be producing the results they are aiming for. Now that I am more independent and running my own experiments, I am beginning to experience the bewilderment of obtaining a result that just isn’t quite right. This has certainly left me scratching my head without a clue of what has gone wrong.

I have been trying to perfect the use of a PhosTag gel: this technique will help me to identify very clearly any proteins in the cell that have been phosphorylated (the addition of a PO32- group) as a result of certain treatments I may give to the cells. As I mentioned in my last blog, I am examining how the phosphorylation of a very specific set of proteins, known as the Rab proteins, changes under certain conditions. For this, PhosTag gels are an invaluable technique, which it is essential I perfect. However, PhosTag gels are proving a little more fiddly than the western blot technique I learnt in my first couple of weeks. I have been struggling to reproduce my results (getting the same result every time I conduct a PhosTag gel) and am struggling to troubleshoot this relatively lengthy process with lots of opportunities for error.

But I have not lost hope. At the moment, I am working with samples I have produced from wild type HeLa cells. HeLa cells are a cancer cell line that are derived from cervical cancer cells taken from Henrietta Lacks (hence, HeLa) in 1951. These cells have an incredible ability to proliferate and are very robust so are used extensively in research as a go-to model. One of the potential stumbling blocks I may be tripping over in my PhosTag gels is the fact the level of Rab protein in HeLa cells is low. The technique is not sufficiently sensitive for me to be able to see proteins that have a very low abundance so one of my problems may be that I simply don’t have enough of the Rab protein to detect. This doesn’t mean that it’s all over! Next week I am hoping to overexpress the specific Rab protein I am examining in the HeLa cells. I will artificially introduce the Rab protein into the cells to increase its abundance in the cell. This will allow me to be able to detect both the Rab protein itself and any phosphorylation of it that may occur much more clearly because there will be more protein there for me to see.

So, all fingers and toes are crossed and hopefully by my next blog I will have achieved a successful PhosTag gel and added a few more experimental techniques to my skill set.