Week 8 - 10

Hello, this is Jacky Jiang from McAlpine group in Princeton. It has been a long time from last post. In the past three weeks, we keep on testing the efficiency of our methods to make thylakoid and the concentration of our chlorophyll.

To get better concentration, I need to come up with different methods of producing the thylakoid. As I have tried the classic procedures, the basic steps would be similar. First of all, I need to use a lab blender to blend the mixture of alginate leaf pieces with grinding buffer. After we get the solution, we will put it into the centrifuge for the pellet. The pellet we get need to go through another step, which is called resuspension. In this step, we put the pellet into the washing buffer and do the resuspension procedure. Then, we need to go through few more times of centrifuge to get the concentrated thylakoid. To make modification, I tried different kinds of centrifuge rate, which made the composition of the pellet different. This change could be very critical. Since the nuclei and other fragments of plant cells have different density, the centrifuge rate determines which component would be at the bottom of the pellet. What’s more, I also change the grinding buffer I used for blending the mixture. The different concentration of tricine would make the grinding level different, so that the size of membrane fragments would be different, too.

After I tried different methods, the results comes out that the classic steps with appropriate concentration of grinding buffer and high centrifuge rate worked the best. I also tried to change the sequence of centrifuging and buffer mixing, which didn’t turn out well in the end.

 To determine if our thylakoid is efficient enough, we still need to determine it by the concentration of chlorophyll. The experiment methods are the same as we did last time. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to 10 mL of 80% 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 50 mL 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.  

Our thylakoid concentration has improved a lot after we modify the methods. In the rest of the summer, we will move on to the electrical part of our project.