UROS 2017 Project: Analysis of the Cellular function of potential new players in Homologous Recombination
By Corinne Woodcock
After receiving my offer of a place on the University of Nottingham BBSRC funded Doctoral training partnership (DTP) scheme, I wanted to seek an opportunity to expand my laboratory skillset prior to starting. After to speaking to Dr Csanad Bachrati about this he made me aware of UROS which I applied for and I was awarded with the funding. My project was titled ‘Analysis of the cellular function of potential new players in homologous recombination’.
Before starting my project, Dr Bachrati had given me directed reading on the techniques that would be involved and gave very supportive supervision during the first few weeks. I started off gaining confidence in cell culture to ensure that the cells were kept healthy and at a confluency that allowed experiments to be carried out.
The first major experiment was GFP-immunoprecipitation which involved using GFP beads which bind the GFP tagged protein within the cells pulling down any interacting bound proteins to the GFP-tagged protein. After harvesting and lysing the cells, I then performed Western Blotting using a candidate based approach when antibody probing to identify any interacting partners involved in homologous recombination. Western blotting involves preparing samples and running a SDS-page gel to separate proteins which are then blotted onto a membrane. I probed the membranes with various antibodies, incubated it with a substrate and detected any signal using a LI-COR imager.
After repeating this several times using different tagged proteins of interest, some potential interacting partners were identified. Next, I performed immunofluorescent staining on the HDX GFP-tagged cells. This was to verify localisation of HDX and that of the stained protein (FANCD2- a known homologous recombination protein). This involved growing the cells on coverslips and inducing the cells to express the GFP. These were then stained with FANCD2 antibody (a known HR protein) and mounted on microslides with DAPI. Using a confocal microscope, the fluorescent coloured cells and staining was visualised, allowing localisation of the proteins to be identified. HDX was found to localise in the nucleus. I also used SiRNA transfection to knockdown the expression of a protein of interest, however, due to time constraints I was not able to optimise this
I also had the chance to attend a seminar hosted by the Lincoln Institute of Health in which Dr Bachrati presented his research, including some of the results that I had gained. My UROS experience has been invaluable in learning techniques and building my confidence within the laboratory prior to embarking on my PhD. Gaining experience making a poster and presenting this at a conference will also be very valuable for the future.
*To view Corinne’s project poster, please click on the thumbnail below