Day Seven

Continuing where we left off ytd, today we continued with western blotting as well as starting on flow cytometry.

For western blotting:

1) The overnight primary antibody incubated membrane blots were washed thrice with tris-buffered saline (TBS), containing 0.1% tween (TBST).
2) Secondary antibodies targeting the specific animal species of the antibody was added. For example: Goat anti-rabbit secondary antibodies were added to detect rabbit derived anti-human tubulin antibodies. While donkey anti-goat antibodies to detect goat derived anti-vimentin antibodies.
3) The cells undergo incubation for 1hour at room temperature.
4) After washing thoroughly with TBST again, the membrance blots were dried in a 37⁰C incubator.
6) We used an scanner to scan the dried membrane.

For flow cytometry, we used the cells we had from ytd. Those with Palitaxel and those without.

1. We washed the cells with PBS for about 3 times
2. Trypsin is added to detach the cells from the plate surface. Weak trypsin is used for MB 231, unlike for RT3 and MCF-7. This is because MB 231 cells are less attached to the plate surface so less trypsin is required. 
3. The excess trypsin is neutralised with PBS or 0.1% fetal bovine serum (FBS). 
4. Centrifuge to spin down the cells, and discard the trypsin carefully.
5. 70% ethanol is added for cell fixation, which preserves the cells in it's life-like state although the cells are already dead. 
6. Wash with PBS with 0.5% BSA. This prevents the clumping of cells, ensuring even dispersing.
7. Hydrochloric acid is added to denature the DNA by disrupting the hydrogen bonding of the DNA duplex, hence unwinding the DNA helix to expose the thymidine analog (BrdU).
8. Cells are washed with PBS with 0.5% BSA before the addition of FITC conjugated anti-BrdU antibodies. FITC is a green fluorescence dye. 
9. Incubate for 1 h at 37⁰C.
10. Propidium iodide (PI), a DNA binding dye, is added to the cells before subjecting to flow cytometry analysis. 

Ivan used this motor to suck up the remaining cell culture medium. COOL.

This box is used to store the membrane with the antibodies during incubation. The antibodies are light-sensitive so they cannot be exposed to light for long.

Having fun pouring the supernatant away after each centrifugation. 

Propidium iodide. It looks like a carrot. 

The machine we used for flow cytometry

Ideal BrdU results. Ours didn't come our ideally. :(

Scanner for western blotting

This is a guide for the protein ladder. The different colours and height represent the varying mass.

Western blotting results for tubulin

Western blotting results for vimentin

We had a great time with Ivan. Thanks, Ivan! (BTW he sat down, so he doesn't look very tall)

So true.

KEHEHEH.

This are some of the other guys who work in the same lab as Ivan. We had a lot of fun in SBS partly because of them. As quoted from Mingjie, they are a "fun-loving bunch". XD

Mingjie and Ivan gave us this one-hour talk about how research career is like. The moral of the story is you must have passion in science if you want to join the research career. Mingjie was discouraging us to join research and Ivan was encouraging us. HEHE. But all of them have a passion for science and that is what matters. 

IVAN IS TALL!!!!!!!!

Its been a while since we posted our own individual views. But I really want to share my experience at SBS. So as explained above, the labs and office in SBS are next to each other. Personally, I prefer SBS to MSE, mainly because of its environment. In the SBS lab, I could feel that there people working inside are pretty close to each other (let's hope I am not wrong). They would help each other to do small small things, like preparing solutions or even buying food. There is friendliness and warmth in that lab and I hope I would get to work in such an environment in the future. I hope I can return there someday again. Hehe.

Mingjie and Ivan also talked to us about how research is like and the pros and cons of it. After talking to them, I could feel the passion they had for science and research and I really admire them for that. I also felt that the one hour spent talking to them was worth it, even though I was starving already. What they said are really their own experiences and they gave me a realistic view on how research is like. It is better than all those talks or open house events. Although I was slightly put off from research from Mingjie's words, I still love science and have no other intention of deviating from it. 

Karmen

Hello! It's time for our individual reflections! Actually, I also share the same sentiments as Karmen. I really like the SBS's labs and the people in there. They helped each other a lot, by helping prepare the other person's solutions (if he really needs it) and joked a lot with each other. It was really a lively place! I also learnt a lot about the different methods used in molecular biology. It was related to the work that we learnt in school. So, I was able to understand the things that Ivan was saying. In the lab, there were really a lot of machines to use and know. They have to know the different computer programmes which are associated with the different machines. It was really amazing! Anyways, the one hour lecture from Mingjie and Ivan was quite inspiring to me, as I had never seen people who had that much passion for their work and projects. They really love the things that they do, even though it may be tough most of the times. Looking at them speak about research with so much passion, I was awed and felt a bit guilty. They not only talk about research, but they also talk about the different areas (mostly, of the cons). It made me think of my future career path and what I would really like to see myself doing in 20 years' time. To know that they are driven mainly by their passion and interest in the fields, it made me feel that the people who are out in the workforce are working on the things that they love, despite the lack of sleep and the many things that they had to read on! So, I really want to thank Ivan and Mingjie for teaching me that valuable lesson.

Alethea

Hi! This is Yuh Harn. i fully agree with what Karmen and Alethea said in their comments. Indeed the environment at SBS is more fun-loving and more informal than in MSE. Random jokes and a helping hand from time to time would be really nice. But for me, perhaps it's just my personality, but i would prefer working at MSE, with a more distraction-free environment when working. A little chat and laughter here and there but when working i would prefer full concentration. Everyone has different preferances, so this is just mine. And it has been great talking to Ivan and MJ about career choices, having to choose uni courses within the next few months can feel stressful and i'm glad to have had a 'prep talk' by them.

Yuh Harn

And that marks the end of our time at SBS. Not only did we had a fun time, we also made new friends with the guys in the lab. They have also became our supporters! We thank them very much for their support and patience with us for the past two days! We wish them the best from here on. 

We will be working with Dr Wen Feng tomorrow back at MSE. Cheers!