Prepping for Peptide Synthesis

Today, (December 13th), I went to RPI for my final research day of the first semester! Today I worked on prepping for another peptide synthesis. We are working to attach a peptide to a new linker that attaches to a resin bead. The concept is similar to another idea that I introduced in a previous post. These beads will eventually be packed into a column and used to purify biologics with the binding of the peptides on the beads.

My first job was to make 25 mL of 0.1 M NaOH from 2 M NaOH. Using the formula (M1)(V1)=(M2)(V2), I found that I would need to add 1.25 mL of 2 M NaOH and fill the rest of the volume with PBS. To carry out the dilution, I used a volumetric flask.

Next, I made 50 mL of 20% ethanol solution. To do this, I added 10 mL of ethanol to 40 mL of distilled water. After I mixed this solution, I then added 150 microliters to each of 20 wells in a well plate. After waiting for half an hour for the ethanol solution to evaporate, I added 150 more microliters to each well.

I can't believe that the first semester of my senior year is already over! I can't wait to see what's in store for me starting in January.

New Microarray Experiment

On Friday (December 6th), I finally returned to RPI! JP explained to me a new experiment that we're running. We are now running a microarray to analyze the binding of HCP proteins that we do not want to bind to the biologic that we are creating. This information will be used in the purification work I described in an earlier post.

Today, most of my time was spent waiting for the microarray slides to incubate (they had to incubate for an entire two hours!). While I waited, Doug and I adjusted the pH of the PBS he was making using a 1M NaOH solution to increase the pH to 5.5.  I then labeled the tubes for HCP and PBS solutions and the petri dishes containing the slides that would eventually be used with those solutions. Some tubes would contain 1 mL of PBS, some would contain 1 mL of HCP solution, and some would contain a mixture of half PBS and half HCP solution. After I finished labeling, I added the indicated amounts of PBS and HCP solution to the tubes.

After the tubes were all filled, Doug and I made amino acid solutions. After some practice, I'm definitely getting faster at weighing out the amino acids!

When the slides were finally done incubating, we washed them three times with the new PBS solution for 10 minutes per wash. By the time the washes were over, it was time for me to go back to Emma!

I can't wait to return to RPI next week for my last visit of the semester and see the results of this experiment!

Scanning Slides

Last Friday (November 22nd), I returned to RPI for more work! Currently, we have three methods of getting the protein that we are focusing on. The first method involves growing the protein in E-coli and lysing the cells. The other two methods involve extracting the protein from human brain cells using different buffers (which I will call buffer R and buffer T). As I have demonstrated in previous posts, our protein consists of two loops that react with each other to form a tight junction.

In all of our previous experiments, we have used only parts of the protein in microarray experiments. Today, we analyzed microarray slides that tested the entirety of the protein for the first time. We used a scanner to analyze slides that tested protein derived from each of the three methods. We scanned nine slides total. 100%, 50%, 10%, and 1% dilution for the E.Coli protein, 50% and 10% dilution for the buffer R protein, and 50% and 10% dilution for the buffer T protein. We scanned each group separately for comparison. With each group, we also scanned a control slide which consisted of no focus protein- only primary antibody- to make sure the antibody was binding specifically to the focus protein. Examples of good and bad binding of the primary antibody are seen below. The blue circles represent focus protein that was bound to the microarray, and the red represents primary antibody. We want the primary antibody to bind only to the focus protein.

For the scan, I laid each slide face down on the scanner, each oriented the same direction. I had to be careful to line them up straight right along the edge of the scanner, so later analysis using spotfinder would be easier to line up the microarray spots. 

An example slide arrangement is seen below for the E.coli slides. 

We scanned the slides first at 200 microns, then at 50 to get the most precise scan possible. After getting the images from the scanner, we imported them into a program called spotfinder to analyze the intensity of each spot. The resulting data wasn't perfect, but it was a good start! 

I will not be able to go to RPI next Friday (November 29th) due to the Thanksgiving holiday, but I look forward to returning on December 6th!