The Xanthophyll Cycle in Photosystem II

Wednesday, 11 June 2014

Third Repeat: Isolation of Stacked Thylakoid Experiment

So this was my third time of repeating the stacked thylakoid protocol, changing the parameters yet again.
We had a sample loop of 300ul for the injection value (smaller the sample loop the greater the resolution of our FPLC curve separation)
Also, we used a higher alpha DM detergent concentration of 1.2%

Due to the fact this was my third time doing this experiment, I was pretty rapid so had more times to take random snaps around the lab. Quite unfortunate as I actually forgot my camera on this day, oh well my camera is pretty decent with its 8megapixels:
One of Ruban's lab, the other one being a dark adapted room!

While waiting for the light treatment to occur on the spinach leaves, Petra made us do calculations to work out the correct mass of each chemical we needed. It was already on the sheet, but it's always good to get practice. I always hated these in lab practicals as they always confused me, but have to say they are not just something the lecturers make you do for nothing! They are extremely useful in the working life of a scientist!

After using the polytron to grind up all the spinach leaves in a uniform liquid, filtering through the layers of muslin; getting rid of the large starch particles in the spinach leaves we do not desire. Ohhh so much green! :3

Always refering back to the lab book and my little written notes! See my pencil case? Very tempted to get a large see through one that can fit in all my highlighters, pen, pencils, calculators, currently everything chucked into my hello kitty pencil makes it a "fat hello kitty" :( Generally don't like the bulkyness in my bag. Also, clear through pencil cases means I don't have to mess around with removing my pens for the exams~ I can just take the whole thing into the dam exam! :)

Everything must be kept on ice!!! Preventing any biochemical reactions from occuring. Oh look the ice even started to turn green! Haha! (I think it was from a minor spillage)

This machine spins the box in a square manner for the amount of time we desired. This is the part which we call the dark treatment. We usually, after setting the spinning speed place a black cloth over our ice box with our material in, to aid the dark conditions, for treatment.

Bag full of micro centrifuge tubes!

Here they are hidden in the draw~

Vivid colours in the lab~ always good to add colour to your life. Especially in the lab its always ever so grey in that room!  Here I am adding up my O/D for chorophyll A and B measurements in the spinach leaves.

Syringe, which we use to load our sample into the 300ul sample loop at the injection valve!

 Then we ran FPLC to separate our fractions! 

However, this time of stacked thylakoid preparation, Erica (The postdoctoral researcher at Ruban's Lab) allowed us to analyse certain fractions we obtained from FPLC via a method called 77k (using a machine called fluoromax-3). This method allowed us to determine exactly what was in our fractions, as before we were estimating from previous graphs as we knew what peaks would give what complexes. Also, it allowed us to see how efficient our preparation in stacked thylakoids were in providing us with our desired PSII super-complexes only. All this was performed in the dark room (Ruban's second lab)!

Always keeping our fractions on ice prevent the reactions !

As you can tell from the illumination from the computer screen, we are in the dark room. The 77k part is the white cylinder that Erica's hand is placed around. She is adjusting the sample to allow the florescence to hit our sample in order to detect what is actually inside!  

Adjusting the programmes parameters to get a clearer scatterpatch which we can use to determine the exact complexes in our fractions.

A black cloth over the 77k part of the fluoromax-3, aiding the conditions even more, even though the machine is in a dark room already and lights are switched off!

The name.
 To conclude, what we found from the 77k is that our A9 fractions which are meant to contain high amounts of PSII supercomplexes, still contain a high amount of PSI supercomplexes. Therefore, we know for sure that the stacked thylakoid preparation is not suitable for what we desire- isolating only PSII supercomplexes so we can analyse the efficiency of Violaxanthin to Zeaxanthin of the xanthophyll cycle, which occurs when the plant is in high light stress.

This leads to the fact we know now to use the BBY protocol, which is a preparation very similar to that of isolation of stacked thylakoids, however much longer as more steps are involved. However, at least with BBY protocol obtaining only PSII super complexes is the standard outcome.

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