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!

Microscopy!

Today (November 8th), we worked with cells from the blood-brain barrier using microscopy! By definition, microscopy is "investigation using a microscope." To refresh my knowledge of the microscopy processes we use, JP gave me this link to read. In our work, we used Alexa 488 and Hoechst 33342 (also known as DAPI). Before I arrived, JP fixed the cells and added the primary antibody. We had to decide whether or not to permeablize the cells before adding the DAPI. We decided to try imaging the cells without permeablization first. We added a DAPI solution to the sample, incubated the cells, and then removed the excess solution. We then washed the sample a couple times to remove excess dye.

Once the sample was ready to be imaged, we had to decide what wavelengths of lasers to use to image them. To do this, we used a program called SpectraViewer. The resulting graphs are shown below.

 The vertical line on the graphs represents the wavelength of the selected laser. The green curve represents the Hoechst 33342 and the red curve represents the Alexa 488. The shaded regions represent the region that will be recorded. We decided to use the 405 nm laser for the Hoechst 33342 and the 488 nm laser for the Alexa 488.

To image the cells, we used a Zeiss Spectral Confocal Microscope (seen in the image below). It was so cool to be able to see the cells magnified to such a high degree!

The nuclei were very clearly visible, but the staining from the Hoechst 33342 was not visible. After viewing the images, we decided that it would be necessary to permeabilize the cells and re-add the Hoechst.

I won't be able to go to RPI next week because I will be in Kansas City for my national championship horse show, but I can't wait to return on the 22nd to see the images from the cells and continue our work!

Analyzing Peptide Mapping Data

On Friday (November 1st), I did a lot of work with analyzing peptide mapping data using the Origin program. But before I get into that, JP said that the peptide formation that I prepped for last week came out perfect! Anyway, I worked with three different sets of data from the same experiment- one set of 20 peptides and two sets of 56. After copying the data from a previous Excel sheet, my first step was to change the settings of all of the odd-numbered columns to "Y-error" because it represented standard deviation data. After I had done that for all three sets of data, I adjusted the fit curves by using a one-site bind pharmacology non-linear cure fit. In that process, I had to change the k-values of every data set to 1 to standardize the fits.

 Our goal is to look at the data for each of the peptides and determine which peptides have low Kd (affinity towards the substrate) values and high R-squared values (good fit to the best-fit line). The area we are looking for is represented by the green area above. In looking at the plots, I narrowed the range to an R-squared value of 0.5 to 1 and Kd value of 3E-6 to 8E-6, and changed the data point labels to display their peptide numbers. Looking at the layout of peptides in this region, we determined that the most accurate set of data is the second set of 56, which makes sense because those peptides were made in 2012, while the peptides used in the other two sets were made in 2011.

After looking at those plots, I the plotted Peptide # vs. 1/Kd. This produced plots with many sporadic troughs and peaks. JP introduced me to the idea of curve-smoothing. Using the Kd values of the peptides, we are ideally looking for the Kd values of individual amino acids. To do this, we use averages of the Kd values of different peptides. For example, A would equal KD1, B would equal the average of KD1 and KD2, etc.

Using an Excel template that JP made for a previous experiment, I plotted averages of three up to fifteen peptides. While the fifteen-peptide averages obviously smoothed the curve the most, it is hard to determine the correct plot we should use because as the curve gets smoother, it hides certain important peaks.

After I finished my analysis work, JP showed me some of the brain cells that he is culturing! He just started growing them on Tuesday, so they had not yet grown into a full layer of cells. They will be used to run a second trial of an experiment we did last winter, which involves testing the permeability of the blood-brain barrier by passing different sizes of sugar molecules through a layer of cells. I can't wait to continue working with the data and see where that experiment goes!

Purification Experiment Set-Up

Last Friday (October 25th), JP introduced me to another project that our lab is working on. Previously, we had two different ways to produce free peptide. First, we could grow peptides off cellulose and dissolve the cellulose in DMSO to leave the free pepide in solution. Second, we could grow peptides off of a polymer bead that has  functional groups for the peptide to attach to. These linkers could then be cut using a strong acid to produce free peptide. Now, we are working on producing peptide that grows directly off of the polymer bead, so it cannot be cleaved. Using these beads in a column, we could purify biologic. When biologic is entered into the top of the column, it would be captured by the column through the peptides. We could then elute off the biologic.

To prep for this experiment, I inserted seven specific amino acid sequences into the peptide machine program, copying each 13 times. After inserting the sequences, I used the program to see what volume of each amino acid prepped amino acids for the peptide machine. I then rounded those values up to either 5, 10, or 15 mL of solution. After determining the amount of each solution we would need, I calculated the gram amount of amino acid and mL amount of NMT to be added to make each solution. I then labeled each amino acid tube and added the specified grams of amino acid to each.

We will use the amino acid solutions that I made today to produce the seven specified peptides that I entered into the program. I look forward to returning to RPI next Friday to see what progress has been made!

HCP Assay

On Friday (October 11th), I had a variety of different tasks at RPI. My main task was to help Doug with an host cell protein (HCP) assay. In this experiment, we plan to analyze the binding of HCP to a peptide array using high-thoroughput screening of fluorescent tags. To do so, the peptide arrays will be incubated with HCP solution, primary antibody, and secondary antibody, separated by thorough washings to remove the excess solution. We will be testing different concentrations of primary antibody. The secondary antibody is fluorescently tagged, so we can screen the slides for intensity, indicating the intensity of HCP bound by each peptide.

Figure by JP Trasatti, Karande Lab, RPI

Previously, Doug printed peptides arrays onto three different slides and coated them with an HCP solution to bind the peptides. My first job was to wash the HCP solution off of the slides. This washing involves pouring off the solution from the slide and adding 10 mL of PBS to the petri dish, then rotating them for 10 minutes to wash off any excess HCP that was not bound by the peptide array. We then made the different concentrations of primary antibody and applied them to the respective slides, making sure all of the solution stayed on the slide and was evenly distributed. After an hour of incubation, we then washed the primary antibody and applied the light-sensitive secondary antibody.

While we were waiting for the primary antibody incubation, we were going to make western transfer buffer, but we did not have enough methanol. Instead, we made 2L of PBS, which is a process I have done before!

I am excited to return to RPI and see the results of this experiment, but I will unfortunately not be able to go this Friday (October 18th) due to Parents' Day.