IF images

IF images

Here are some of the images I obtained during the summer with the confocal microscope. These are HeLa cells exposed to different conditions in order to see how different factors affect localization of CHIP.  The table in the word doc shows different channels collected by different filters of the confocal microscope after excitation of sample with photons. It was really cool to be able to learn so much from this technique. The images were then analyzed via Fiji image processing program. I was able to combine these channels to illustrate merged image. Obtaining images was probably the hardest part and  just optimizing conditions for images. I look forward to learning more about using the microscope in order to help me collect data in the future. After my experiment was over, I gave an oral presentation to two groups at MPI at one of the meetings. It was great to get up there and tell them about my work.  I look forward to sharing my work at W&M soon.

Conclusions

I wanted to post some images of my IF experiment. The computer is not liking that too much so I will just save that for later in September for my poster presentation. Instead here are my conclusion and what was accomplished during this study. THANK YOU. I have learned so much and this opportunity without a doubt will help me in my future career as a biomedical scientist.

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THE END?

Basically the following describes what I did over the summer. In addition to this summary, I learned how to do the entire Western Blot process and got introduced to chIP process. It was an unforgettable summer with so much skill learning. It’s unreal how much I benefited. I know I will carry these invaluable skills with me for the rest of my life especially as I look forward to conducting research in medicine and diseases. COULD NOT BE MORE GRATEFUL for what I have received… I will make it count. Thank you.

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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.