Blocking Buffer Project Introduction

On Friday (January 24th), I worked on planning my project for the Spring semester. I will be testing concentrations of blocking buffers and the time and temperature used for blocking. In my experiments, I will be testing two popular blocking buffers: Bovine Serum Albumin (BSA) and Casein. In my research last week, I found that the concentrations of BSA used in research procedures are usually between 0.2% and 5%, and concentrations of Casein are usually between 1/2x and 1x. Using this information, I decided on a project plan that analyzes 0.2, 1, 2.5, and 5% concentrations of BSA and 1/2x and 1x concentrations of Casein. I will be testing the BSA concentrations at room temperature for blocking times of 30 minutes, 1 hour, and 2 hours, and at 4˚C blocked overnight. I will be testing the Casein concentrations at room temperature and 37˚C for blocking times of 30 minutes, 1 hour, and 2 hours, and at 4˚C overnight. Below shows my initial tables for the project. The last table indicates how many slides I will need for each temperature for each blocking buffer. 

Overall, my goal is to determine the least amount of blocking buffer and the least amount of blocking time that will be effective in blocking the slides. I will collect quantitative data by examining the intensity of fluorescence that results on each slide when each slide is coated with SYPRO dye. Last week, I determined that the SYPRO dye has a pH of 5. This acidity could cause the SYPRO dye to elute protein off of the slides. Ideally, we would like to raise the pH of the dye to 7.4. However, raising the pH can cause the dye to fall out of solution and form a solid. 

Today, I tested the pH that I could raise the SYPRO dye to before it fell out of the solution. To do this, I tested a 1 mL sample of the dye, adding 1 µL of 2M NaOH at a time and spinning the sample down for 1 minutes to observe if any solid formed.

Once I observed a clear, jellylike solid substance forming from the solution, I used pH strips to test the pH of the solution, which I found to be around 6. I then continued to raise the pH of the dye to pH 10 to see if there were any other effects of increasing the pH. I found that there were no effects other than a clear, jelly-like substance forming at the bottom of the sample. We then wanted to know if any of the dye stayed in the solution. To do this, I took small samples of the original dye, the supernatant that was left after I spun down the sample, and the jelly-like substance. JP and I then observed these samples under UV light to observe the fluorescence of the dye. We found that both the jelly-like substance and the supernatant were about half as fluorescent as the original dye.

I can't wait to continue developing the details of my project next week!