Resource | Mollie's Blog No 8
Meet the Scientists
Happy New Year!
Prior to Christmas I left the lab knowing that there was a mechanism in cells that reverses the Ubiquitin phosphorylation brought about by the activation of the PINK1/Parkin pathway. With the new year ahead I am now looking to investigate whether my protein of interest, PPP2R5D, is involved in this removal of phosphates.
In order to work with a protein, it is critical that you have a reliable method of detecting it. In the lab we use antibodies that have been generated to detect a specific protein – this allows us to ‘see’ the protein we are looking for clearly amongst the thousands, if not hundreds of thousands, of other proteins within our samples. However, sometimes the antibodies are not very specific (they will detect other proteins in the sample) or require extremely high levels of the protein in the cells to visualise it. Therefore, my first task is to determine how good my antibody is at detecting PPP2R5D.
To do this I will overexpress PPP2R5D in cells. This means I will artificially introduce copies of the PPP2R5D gene into cells by a process known as transient transfection. Transient transfection will result in substantially higher levels of the PPP2R5D protein in the cells than there would be naturally. It is important to note here the ‘transient’ nature of this process. The PPP2R5D gene I add to the cells will not become integrated into their genome. This means that after just a few days the cells will return to their normal levels of PPP2R5D as the extra genetic material will be lost during cell division.
The best way to establish whether the protein that my PPP2R5D antibody is detecting is truly PPP2R5D, I will use a reporter gene. What is this? Attached to the end of the PPP2R5D gene that I will add to the cells is a gene for Green Fluorescent Protein (or GFP) – this is my reporter gene. When the cells produce the PPP2R5D protein from the gene I add, the protein will also have the GFP attached to it. We have very good antibodies against GFP so, theoretically, if the GFP antibodies detect the same proteins as indicated by my PPP2R5D antibody, that should tell me they are both detecting the same protein and that that protein is PPP2R5D.
Another very interesting use of reporter genes is in immunofluorescence.
This is a technique in which you attach various fluorescent proteins to other proteins whose cellular location you wish to visualise, or even track their movements in the cell. This produces some very beautiful images, such as the one of neurons in the brain to the right. This can even be scaled up to look at whole organisms such as the turtle embryo below.
This image became famous last year when it became a winner of the Nikon Small World photomicrography contest. Sadly, I will not be producing anything as spectacular as either of these images, however, I do hope to use this technique to observe whether the PPP2R5D protein moves around the cell.
I feel very fortunate to have been given the opportunity to continue my research in these challenging times and extend my very best of wishes to everyone. Let us hope we are able to turn a corner soon and make 2021 a happier year for all.