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!
Great post! I love the technical detail, and the sense of trial and error.
ReplyDeleteCould you please include a sentence in each post that relates your daily work to your broader project, when possible?
Kailin, you've obviously been doing this internship for a long time so it's very difficult for me to follow exactly what you do. Even though you already explained it to me in person, many of the details are still extremely vague. However, from what I do understand, I'm really impressed by the meticulousness and careful detail that your internship necessitates. The visuals you put into your blog posts are also really impressive and it definitely helps me understand what you're doing a little more!
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