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.

Host Cell Proteins

On Friday (October 4th), I finally returned to RPI after missing a week! I worked with Doug (one of the undergraduates in my lab) on a project he is working on involving host cell proteins. A couple weeks ago, these host cell proteins were involved in the SDS-PAGE gel we were working on!

Previously, we have only had intensity data to analyze the amount of host cell protein (HCP) that is bound by different peptides. This is relative data, so it does not give us information about the actual amount of HCP that binds. Ideally, we want to find a peptide that has a high affinity for the target protein we are looking to purify, but low intensity of HCP (the green square in the graph below). We do not want the result to be in the red square, indicating high affinity for the target protein, but high intensity of HCP.

Doug is working to quantify the amount of HCP that is bound by the peptides. To do so, he is printing different concentrations of HCP on nitrocellulose (negatively charged paper) 3X5 microarrays. These spots of known HCP amount will then be analyzed for intensity to determine a standard curve. The standard curve will then be used to determine the unknown (amount of HCP) for the peptide data.

My first job was to pipette 80 microliters of 6 different concentrations of HCP into their specified positions in the printer well-plate. We then cut the nitrocellulose paper into slide-shaped pieces and taped them onto the printer so they wouldn't move during printing. When we were setting the heights for the printer needle, one of the pieces of nitrocellulose cracked, so we had to untape and redo the nitrocellulose. Once we finally had the printer set up, we set the first round of printing to run, and we found that the middle spot on the second slide was not printing. We then reconfigured the needle heights and made each height tighter to the nitrocellulose. As we continued to run the machine, we realized that the needle was popping up every time it went to print on the first slide, so we had to tap the needle down every time it was positioned on the first slide. Because the printing was taking so long, we decided to only do 10 runs instead of the original 20 runs planned. Even the 10 runs took over 2 hours to complete!

I look forward to returning to RPI next week to see what data they collected from this experiment!

More Amino Acids

Last Friday (September 20th), I returned to RPI for more lab work. I had a shorter day today because I had to leave early to go to Massachusetts for a horse show. This week, I continued to work on preparing amino acid solutions. I finished the rest of the 20 amino acids that I started to make last week! These amino acid solutions will eventually be used in microarray experiments.

I will not be able to go to RPI next Friday because I am participating in a program at Cornell, but I'll look forward to returning on October 4th!!

Data Preparation and Amino Acids

Yesterday (September 13th), I went over to RPI for more work in the lab. I got to see the gel I prepared last week! Unfortunately, the gel turned out useless because it had too many metal ions which caused there to be what was basically an extended row of dye instead of separated bars. The gel was still helpful, though, because now we will not attempt any more gels with those types of solutions!

My first job for the day was to compile some of the data from our previous Claudin-5 microarrays. For each of our past experiments, we have pixel intensity data for the resulting microarrays. At this point, we want to compile all of the past data to be able to look at it as a whole. On JP's computer, each past experiment has it's own folder, so I copied an .mev converter file into each folder. After doing so, I opened the .mev converter file and all of the .mev files for a certain experiment, and I moved all of the data into the converter file. After doing so for every experiment, the .mev converters were ready to run, and they will be run by next week!

My second job was to prepare tubes to make amino acid solutions. I labeled each tube:
Three-letter code (one-letter code)
Mg amino acid to be added
mL solution to be added

After doing this for all 20 amino acids, I then began to add the specified amount of each amino acid to its specified tube.

I can't wait to continue my research next Friday!!

Preparation for an SDS-PAGE Gel

Last Friday (September 6th), I went over to RPI for the first time during this school year! My primary job for the day was preparing for an SDS-PAGE gel. For this gel, we are working with a specific protein of interest.Ultimately, the gel will analyze the bands of the null solution (which does not contain the protein of interest, but contains other host cell proteins (HCPs)) versus the bands of the solution that contains the protein of interest (in addition to the other HCPs). To do this, we are working with different types of protein solutions. First, there are the null solution and the protein of interest solution which are both prepared at 1X and 10X concentration. The different concentrations will ultimately lead to different width bands. Also, there are 10X desalt solutions for both the null and protein of interest. In the original solution, the buffer contains metal ions that can affect how the bands are displayed in the gel, making them appear blurred. In the desalt solutions, the buffer has been exchanged to PBS to eliminate the metal ions and get cleaner bands. Once the gel is run, we expect to see something like the simplified gel below. We expect to get certain bands from the null solution that represent the bands of the HCPs, and we expect to get those same bands from the protein of interest with the addition of an extra band that represents the protein of interest (represented in red in the image).

Today, to prepare for running this gel, I first made the Tris/Glycine/SDS buffer by combining the stock solution with water, mixing the buffer, then putting it on ice to keep it cold (keeping the buffer cold allows for sharper bands in the gel). Next, I made the blue Laemmli sample buffer by combining the stock solution with β-mercaptoethanol (BME). I then labled the solution tubes with the different solutions and added 20 micro-liters of Laemmli buffer to each sample. After each sample was allocated into its respective tube, I denatured the protein solutions in a heat bath for one hour. To prevent the sample tubes from popping open in the heat bath, I wrapped each sample with parafilm (which ultimately caused them to stick together). After the proteins were denatured, I spun the samples, and set them aside to cool. I then set up the gel equipment.

The gel was then ready for JP to run later in the day! I can't wait to see the results when I return to RPI on Friday!

Back to Senior Year!

Although I did not blog this summer, I continued to work in the lab at RPI! Over the summer, I learned so many skills that I can't wait to apply to my work this school year. After my experience at RPI last year, I could not be more excited to start another year in the same lab. Spending an extended period of time in the lab has allowed me to continue to practice different skills with the machinery and materials in the lab, so I am now capable of doing much more than when I first started last Fall!  This year, I hope to keep perfecting my skills and to learn new ones. I hope to continue to get a solid feel of what it's really like to work in a college lab before I head off to college myself next year!