For the past couple of weeks I have not been only doing experiments but also learning why they are done a certain way, might seem simple but its a lot of research and understanding. It’s constant questioning of WHY this? WHY that? Always why, why, why. I love it. I love understanding and it is very thrilling to understand. Why do we use Heat Shock? Why not a different stress on the cell? Why is it important? How is this going to help cure diseases? Why do we wash 3 times? Why do we use PBS? Why do we use PFA instead of methanol? My goal was to know the Immunofluorescence abcam procedure like the back of my hand and not only know it but completely understand it. For example for the first step, I know that for the Immunofluorescence protocol we fix the HeLa cells with freshly made Para formaldehyde (PFA) because PFA crosslinks proteins by forming covalent bonds between proteins and anchoring proteins to cytoskeleton but does not compromise the tertiary structure of proteins ( which is what we want). Another commonly used fixing agent is methanol. Why do we use PFA instead? Well, since we are interested in proteins then methanol and similar products would not be optimal in this study since methanol disrupts hydrophobic interactions causing aggregation of proteins. Furthermore already prepared solutions of PFA would most likely contain methanol since it is used to stabilize formaldehyde by preventing oxidation. Why do we use 2% solution in PBS? What is PBS? PBS is an isotonic detergent that stands for phosphate buffered solution it is commonly used since it does not damage cells, cells do not shrink or burst.
This is only understanding the first step of the entire procedure completely!!! It is crazy. I really enjoy it though, so much chemistry and biology. It has been great and I love to be challenged with this question of WHY. Furthermore for the first step I need to know how to prepare a 2% solution something I had not had much experience with until now… These solutions are not ready for use like they were in chemistry lab or in biology lab… You have to make them yourself. Although challenging at first it is one of the most useful skills that I have learned and without a doubt will help me as a future scientist.