Week 5 to 7

Hi, this is Jacky and I am working in McAlpine Lab at Princeton. 

These three weeks have been very busy. Our projects have been moving to the first critical point, which is about measuring the quality of the thylakoids we make.

                The first test we perform is chlorophyll concentration measurement. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to acetone in a test tube. This solution is mixed by inverting several times and then filtered through a Whatman filter paper into a large cuvette using a glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation. Once the chlorophyll concentration is determined, the total chlorophyll yield should be determined by multiply the chlorophyll concentration in mg /mL times the volume (mL). Once the chlorophyll concentration and the total chlorophyll yield is known, the chlorophyll concentration should be adjusted by adding the appropriate amount of Washing Buffer or by centrifuging again the thylakoids and resuspending in the appropriate amount of Washing Buffer.

                By determining the concentration of chlorophyll we make, we can decide if the procedures we use is appropriate since there are a lot of different methods that we are able to choose from literature. It is always important in science research to try as much methods as possible.

                                          

                What’s more, we need to test the efficiency of the thylakoids we extract. In this case, we need to use a chemical method called Hill Reaction. As we learned in Biology class, during light reaction photosynthesis, electrons will end up in NADPH. In Hill reaction, we need to separate thylakoid with stroma so that NADP won’t be available. Instead, we will put DCPIP, a blue oxidant, into the solution. DCPIP is blue in its oxidized form, and becomes colorless when it is reduced during the Hill reaction. Thus, the rate at which electron transport occurs in the Hill reaction can be measured spectrophotometrically (at 620 nm) by following the change in absorbance of DCPIP as it accepts electrons from the electron transport chain. To perform the Hill reaction, a sample of a chloroplast suspension will be mixed with the Hill reaction buffer (containing DCPIP) and exposed to light for a series of 30 second intervals. After each exposure period, the absorbance of the DCPIP will be measured. The absorbance values can then be plotted versus time to determine the rate of DCPIP reduction as a measure of PET.

It turns out that the thylakoids we make are pretty efficient. We will keep on testing and trying for next couple days and shift our focus to other aspects.