The Highs are worth the Lows

My first Western Blot ever. I am thinking of framing it. I was so proud of myself. I did everything from scratch. I know it may seem simple and everyone in the biology world might know how to do it, but it was my first and it was a big thing for me. I casted the stacking and the resolving gel. Again I have learned the WHY of everything. I now know the importance of the pH difference between the stalking and the resolving gel and the importance of the amount of polyacrylamide one adds (depends on the size of the protein of interest). If you study a big protein then you add less polyacrylamide in order for the big proteins to run faster down the gel. The Ammonia Persulphate Solution (APS) added to preparation of gel acts as an initiator and the TEMED as a catalyst so you add those two components last in order to begin polymerization of your gel (not before or it becomes a solid before you can cast it). I heated my samples with loading buffer which contained SDS and loaded it with marker and previously collected samples. I covered everything with running buffer at working conditions (1x this term is also new to me). Then transferred my proteins from the gel to a membrane via current again. Once my proteins were in the membrane, I blocked the membrane with milk, yeah milk, inorder to prevent unspecific binding of the primary antibody and then added the diluted rabbit antibody overnight which was specific to protein of interest which was CHIP in this case. You have to pay attention to where your proteins are at all times and having a marker with protein sizes helps. CHIP was around 35kDa. Then I had to come in the next morning to do washes and place secondary antibody which was anti-rabbit. This secondary antibody is special in that it has Horse Radish Peroxidase. It is used to locate the protein of interest since it is an enzyme that provides a detectable signal. In order to obtain this signal you have to go down to the dark room just like photography to develop film. In the dark room you provide the enzyme with substrate and then it turns it into product that can be detected with the film. It is the coolest thing. I have also used another technique where you can scan the membrane and it detects fluorescence but here you have to use a different secondary antibody, I do not know the specifics just yet, but it’s easily done which I like aswell.
I am telling you all this not because I want to bore you, but to show you how much I have learned… I do not want to simply to tell you that I have learned a lot… I want you to believe me! The more I think about it, the more grateful I am for this experience. It was extremely hands on. It showed me to think and to practice. Yeah it was a lot of mistakes. So many, but that’s what really makes me appreciate it. I am so happy. This has totally been worth it, every minute.

WHY?

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.

Field Work

Day 1:  We arrive at our hotel in Morogoro.  It’s 8000 shillings a night (US$5) and I may be the first white guest they’ve ever had.  We drop our things in the rooms and debrief:

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Week IX

This week has focused on cytokine production and specialized activation of T cell subtypes included in my model (Th17, Th1, Th2) as well as microglia and CNS invasive CD11+ expressing cells like macrophages and dendritic cells. There are many involved and I won’t go into the specifics of what produces what and where they go, only that several pool into the same compartment and add to one amount. Main cytokine circulation occurs in the CNS and lymph node (LN). PGE2 serves a prominent loe in the activation and feedback regulation of IL-23 and IL-17 for TH1 and Th2 activity. I have T cells being activated and interacting with B cells and naturally in-circulating DCs and macrophages. PrPSc enters the LN after being taken up in an unclear method by M cells from the intestinal lumen like other pathogen, and then cross infect cells in Peyer’e Patches that regular populate them and then go into draining LNs. Either by uptake of infection, DCs and macrophages become prion carriers, once in the lymph node they can interact with B cells or directly within germinal centers (which may be preformed by a pre-existing infection). Follicular dendritic cells which highly express PrPC become proliferation sites for the scrapie protein, cross infecting T cells, which then recirculate into the blood and to new sites of injury with a pro-inflammatory goal. If T cells are recruited into the CNS in response to damaged neurons they will cross the BBB barrier, otherwise it seems unlikely that the T cells will enter the CNS enough if at all to infect cells there. Therefore, in pre-existing CNS damage is not occurring, how does the prion enter the brain to trigger more damage and more frequent neuroinvasion of cytotoxic and regulatory cells all potentially cross-infected with prions. This has yet to be elucidated.

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Week VIII

This week has been a difficult, but has produced a new positive direction for the proliferation components of my model. Early in my research I came across antigen presentation to immune cells as a quintessential component of inflammation. DCs and macrophages can act as APCs (antigen presenting cells) and as well as being able to carry PrPSc in their regular circulation. I dismissed the subject because I found no evidence of PrPC or PrPSc acting as anitgens or that antibodies were formed for their ligation. I saw no dire reason to include dynamic interactions with T cell (and several subtypes) if there was nothing to engage their specific immune response. I was wrong. Though DCs through induced maturation process do migrated between the blood, brain, and lymphodial system, they do not directly interact with the non-migratory FDCs in the spleen and lymph node B cell follicles/germinal systems. I have pinpointed FDCs as key spots for PrPSc accumulation and conversion after peripheral infection, but it remained unclear how they left these areas except that neuroinvasion was seem after a certain critical titer load of the scrapie protein. So I looked at what comes in contact with FDCs, turns out B cells and naïve T cells circulate into these areas, and likes a game of tragic telephone can transfer the scrapie protein between them. T cell response can be induced by a prion infection, become primed in the LN or spleen and then reactivated in the perivascular space following cytokine and growth factor mediated TEM of the CSF and BBB. Macrophages and phagocytic DC act as APCs for the reactivation of migrated T cells and then relocalized to neurons damaged by a previous viral infection. A previous infection may not be necessary for prion proliferation because inflammation could be an indirect result of PrP loss-of-function, but an induced specific immune response in concatenation with peripheral prion infection could significantly increase the severity to the disease.

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