Welcome to my blog !
For my PhD project, I have moved to Madrid in Spain. Easily for me, because I come from the South of France (Toulouse), I can travel between my new destination and home in only 50 minutes of flight. I know I am very lucky if I feel homesick ! My host institution is “El Centro Nacional de Biotecnología” and the lab is located in the microbial biotecnology department. The team has been directly very welcoming and the working atmosphere is definitively very nice. Indeed, Spanish people are talkative, at the beginning It was quite complicated to understand everything in Spanish because I didn’t practice since high school but now we can say It is becoming better and better !
Goal of ESR4 project
To come back to the team, the group is studying two “model” intracellular bacterial pathogens important in human and animal health, Listeria monocytogenes and Salmonella enterica serovar Typhimurium. As part of the PATHSENSE network, the goal of my project is to investigate the role of the Listeria stressosome during intracellular pathogenesis. More precisely, we are trying to determine how Listeria regulates the production of stressosome proteins in different stress conditions in both laboratory media (extracellular bacteria) and infection (intracellular bacteria) conditions. To describe the protein composition of the stressosome we are using specific antibodies. In this way, the first part of my project consisted in purifying antibodies from serum of immunized rabbits and this is what I have decided to talk about today!
Antigen-specific affinity purification
We decided to purify antibodies by antigen-specific affinity, that means to first purify the antigens (recombinant protein expressed in E. coli) that will then allow the purification of antibodies. I can say that I will remember for a long time how to purify proteins because at this stage I have purified 11 recombinant proteins and 7 antibodies. Indeed, I want to look at the expression of 12 proteins in total (the stressosome complex and the other proteins involved in the signal transduction cascade that leads to sigB activation). My PI was even worried that I was dreaming of purification columns !!
Here are the columns that I have prepared : for each one the resin (white) is coupled to a defined stressosome protein.
After rabbit immunization by intradermal injection of the antigen (in this case with RsbT protein), serums have been collected at day 53, 74 and 81. The serum contains polyclonal antibodies with specificity to many different antigens or even to different epitopes in the antigen molecule, that is why purifying them will allow to get the highest specificity and sensitivity.
I have purified independently the three serums (D53, D74 and D81). To tell the truth, at the beginning, I was thinking that the serum at D81 will be the most suitable sample to purify antibodies. Indeed at D81, the immune system of the rabbit had more time to produce antibodies against the antigen. Finally it turned out that there is no difference between the three samples.
Here is a result of Western-Blot : I wanted to detect RsbT protein for three different conditions in 2 compartments of Listeria : cytosol and membrane. I am very satisfied with the purification of the antibodies against RsbT : I detect only one band corresponding to the protein !!
Here it is the end of my first post ! Thanks to the antibodies that I have purified I could study the production of stressosome proteins in response to stress and I am going to Kinsale in Ireland next week to present my preliminary results !!
To be continued….