Today I helped with Yaamini and Laura’s DNR sampling.
We started out the day by halving tissue (I worked on Laura’s Geoduck, while Yaamini worked on her oysters) in order to save some for future use if need be.
The samples then went through sonication after they had ____ added and were vortexed.
The samples I used today were:
The plan for the week:
This is what Grace and I did today:
Table 1. Proteins to extract, based on table from a previous lab notebook entry.
| Site | Condition | 1 | 2 | 3 |
|---|---|---|---|---|
| PG | B | N/A | O51 | O52 |
| PG | E | O78 | O56 | O30 |
| FB | B | O43 | O40 | O35 |
| FB | E | N/A | O24 | O49 |
| WB | B | N/A | O121 | O122 |
| WB | E | N/A | O131 | O144 |
| SK | B | O99 | O96 | O113 |
| SK | E | O106 | O102 | O91 |
| CI | B | N/A | O21 | O22 |
| CI | E | O10 | O06 | O04 |
Protocol used can be found here, or viewed below. This solution must be used no later than 24 hours after it is made, or it is no longer viable.
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If there were a pipetting Olympics, I wouldn’t come in last. But I wouldn’t come in first either, or even medal. This was slightly problematic when pipetting every few minutes for the trypsin digestion. It took a while, but we finished!
Last time we performed a digestion, our quantities were calculated based on having 30 µg protein/100 µL. This time, we had 100 µg protein/100 µL. Therefore, we used the quantities of reagents specified in the original protocol.
The first thing Jose and I did were set up heating blocks at 37ºC. Because the heating block in the Roberts Lab did not have the capacity to hold all of our samples, Jose brought one from his lab. We placed additional thermometers in the heating blocks to ensure the temperature was what we wanted.
Based on my calculations for Plate 1 and Plate 2, I pipetted the correct amount of sample and 50mM NH4HCO3 + 6M urea into newly labelled tubes (ex. OBLNK D). Then, I added 6.6 µL of 1.5 M Tris pH 8.8 and 2.2 200 mM TCEP and vortexed the tubes. For our 51 samples, we needed 2-200 µL aliquots of TCEP from the -80ºC freezer. At this point, I went to class. Jose tested the pH of our samples to ensure the pH was still basic, than began the digestion in the heating blocks for one hour.
Figure 1. pH strips for samples. The blue color of the strips indicates the sample pH was still basic.
Figure 2. First heating block set-up.
Figure 3. Temperature confirmation for first heating block.
Figure 4. Second heating block set-up.
Figure 5. Temperature confirmation for second heating block.
After the digestion, I got 6-200 µL of IAA from the -80ºC freezer and covered them with aluminum foil. We added 20µL of IAA to each sample and vortexed the tubes. We covered all of our samples with aluminum foil and let them digest for one hour. Halfway through the digestion set-up, I left for Merril’s Ph.D defense. When I was gone, Jose added 20 µL of DTT to each of our samples, vortexed, and started the digestion. I got back in time to add 1.65 µL of LysC to each sample and started the one hour digestion.
Because Jose needed to leave early, he prepared 25 mM NH4HCO3 solution for all of our samples. After the digestion was over, I added 800 µL of 25 mM NH4HCO3 and 200 µL of HPLC grade methanol to each sample and vortexed them. I then needed to add 3.3 µL of Trypsin to each sample, meaning I needed 9 bottles of 20 µg Trypsin. We only had five bottles, so I needed to borrow 4 bottles from Genome Sciences.
Before using each bottle, I added 20 µL of nanopure water and vortexed. This made a 20 µg/20 µL solution, allowing me to add 3.3 µL of Trypsin to six samples/bottle. After adding Tryspin to all oyster and geoduck samples, I vortexed the tubes and let them sit overnight. I started the digestion at 4 p.m. on Friday evening, and stopped it at 10 a.m. Saturday morning.
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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.
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Roberts Lab 