We have lots of protein!
Using the protocol we generated in December, Jose and I quantified the amount of protein in my oyster samples and Laura’s geoduck samples. Because we used 100 µL 50 mM NH4HCO3 + 6M urea this time, our protein concentrations were generally much higher.
The first thing we did was prepare reagents. Based on the recipes in Rhonda’s original protocol, we made enough 30 mL of 50 mM NH4HCO3 and 6.6 mL of Lysis Buffer. We also made one set of standard solution vials.
Because there were 51 samples total including blanks, we needed three separate microplates.
Figure 1. Microplate arrangement.
To make each plate, Jose first pipetted each standard in triplicate in the first microplate. I added 22 µL of 50 mM NH4HCO3 to each sample vial and vortexed the solution. Jose then pipetted 10 µL of the sample vial contents into the corresponding microplate wells. The only sample that was not run in triplicate was GBLNK. At this point, I needed to get to class. We covered each micrplate with parafilm and placed it in the fridge. The plates were in the fridge from about 9:30 a.m./10:00 a.m. until 1:30 p.m. Right before I came back from class, Jose made the BCA Working Reagent and used a multichannel pipet to add 200 µL of the reagent to each microplate well.
Figure 2. Microplate 1.
Figure 3. Microplate 2.
Figure 4. Microplate 3.
We took our completed microplates to the Genome Sciences Building. Microplate 3 was placed in the Varioskan Flash plate reader. It was incubated at 37 ºC for 30 minutes, shaken, then read at 562 nm. While microplate 3 ran, I incubated Microplate 2 in a separate plate incubator, similar to the one used in December. I kept it in there for 30 minutes at 37 ºC. When it was finished, I took it out and placed Microplate 1 in the incubator. Microplate 2 was out for five minutes before I could place it in the plate reader. In the Varioskan Flash plate reader, Microplate 2 was shaken, then read at 562 nm. Microplate 1 was also incubated at 37ºC for 30 minutes. The incubation period finished after Microplate 2 was read, so Microplate 1 went directly from the incubator to the plate reader. After being shaken, it was read at 562 nm. I sent Laura the data she needed for calculations and kept the data I needed.
Here’s what I used to calculate the concentration of protein in each well: Plate 1 data
I used formulas from my previous calculations to make spreadsheets to analyze my BCA data. The final results can be found below, including the amount of protein and 50 mM NH4HCO3 + 6M urea needed for trypsin digestion.