Platelets are highly specialized blood cells involved in the clotting of blood and vascular repair when we gain an injury. These cells are activated by certain factors, which have effects on the signalling pathways within the cell. Platelets undergo morphological changes which involves cytoskeletal remodeling, in order for them to cover the endothelium of vessels and therefore stop bleeding. We want to study the effects of small G proteins on cytoskeleton dynamics in platelets.
For my UROP (Undergraduate Research Opportunities Programme) Placement, I have been working in the ICMR (Institute of Cardiovascular and Metabolic Research), studying the small G proteins Rap, Rac and Rho, and how their signalling affects cytoskeleton dynamics in platelets.
I have been working with Dr Lucia Stefanini , who has taught me all the necessary skills and techniques that are involved in this lab based work. For example I have learnt how to isolate platelets from blood, western blotting, PCR (Polymerase Chain Reaction), immunoblotting and the method of how to transform bacteria and induce them to start producing a desired protein.
This last method involving bacteria (we used E. coli) was the basis for the first part of our experiment. We induced the bacteria to produce 3 proteins that bind active Rap, Rho and Rac, respectively and we purified the proteins by binding them to beads. In order to visualise this, we firstly ran an SDS-page gel, which seperates proteins based on their molecular weight, then stained with coomasie in order to see the band of our protein.
We were able to express and purify the proteins that bind active Rap and Rac very well. However the protein that binds active Rho proved quite difficult to isolate. We had a few different theories as to why this was. We think that the protein unfolded and precipitated during the induction step so now we are testing different experimental conditions (lower temperature).
The second part of my placement will involve more work with human platelets to measure active Rap, Rac and Rho with the proteins we have purified. Active Rap, Rac and Rho will be visualised buy immunoblotting and fluorescent imaging with antibodies that specifically bind our proteins of interest tagged with fluorescent dyes.
I primarily applied for this placement as Lucia’s Research in platelets is exactly what I have become most interested in so far in my degree, but I was unsure as to whether I wanted to go into Research. However now, just three weeks in I have been given a real insight into how much independence and self satisfaction it gives and would definitely like to pursue a career in Research.