12.11.12
Last Tuesday (December 11th), I went over to RPI for another day of lab work! Today's work was preparing for a new experiment that is analyzing the size of particles that can normally pass through the blood brain barrier. As seen in the figure below, the set up for this experiment will be two wells (one inside the other) with a filter on the bottom of the inner one separating the two. On this filter, we will grow a monolayer of brain cells to represent the blood brain barrier.
We will have many of these setups in order to test solutions with different size molecules. We will be testing solutions with molecules that are 4 kDa, 10 kDa, 20 kDa, 40 kDa, and 70 kDa. All of these molecules are dextrans, or different size sugar molecules. For the normal type blood brain barrier, the permeability to size curve would look like the black line below. With the peptide modulator we are currently researching to find, we hope to make the curve look like the red line below.
In preparation, I prepared two Liters of phosphate buffered saline solution. This PBS can be used to dialyze protein solutions. To make this PBS, I combined 15.52 mL of 1M K
2HPO
4, 4.48 mL of 1M KH
2PO
4,
2 L of distilled water, and 11.6 g of NaCl. After mixing these contents, I had to raise the pH to 7.4 using 1M NaOH base. After many trials and test with a pH probe, the pH reached 7.4, and my PBS was ready to be filtered. To filter the solution, I used a bottle-top filter with a vacuum attached, like the one shown below.
My second job was to label the test tubes for the experiment. 10 mL samples will be taken from the bottom chamber 12 times over a 9 hour period. I labeled the test tubes needed for the first 5 data collections by writing the time on the cap and the time- kDa of the solution on the side of each tiny test tube.
I can't wait until next week to hear the results of the experiment!
Your work is so interesting! The idea of running molecules through a monolayer of cells seems super cool!
ReplyDeleteIn future posts I would like to learn more about peptide modulators. What are they and how do you think they work?
I am impressed with your chemistry skills. Your chem teacher would be proud!
Kailin, did you go to RPI on 18 December? Either way, please blog about that day.
ReplyDeleteKailin,
ReplyDeleteExploring the blood brain barrier sounds fascinating. What is the ultimate goal? Are you trying to figure out molecular size so medicines can be manufactured to slip through to the brain? Or, is it something else? Do they expect that the size is a lot different than those that diffuse through membranes in the rest of the body?
Can you help me understand the peptide modulator? Is sounds like you are using that to learn about the dextrans (which are sugars)--correct? Will you be using a machine that is normally for proteins to study the sugars?
Your diagrams and photos really help me picture what you are doing! Thanks.
I look forward to seeing the single layer of cells! I wonder how you will regulate its growth. Can't wait for your next entry.
Ms. Maier
We are trying to find a peptide modulator molecule that will interfere with the tight junction of claudin-5 protein between the endothelial cells in the blood-brain barrier. This junction prevents the passage of larger medicine molecules through the barrier, and if we can find a way to inhibit the junction, it would allow larger medicine molecules to pass through the barrier. This experiment with the dextran molecules is a preliminary test to find out what sizes of molecules can pass through the uninhibited tight junctions. This data will eventually be used to compare to similar data using different peptide modulators.
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