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Six boxes of trypsin arrived yesterday. Guess what that means?!
Quick tangent: my schedule shifted around a bit, so here’s an updated extraction schedule. I want to finish all of my extraction work before I work from California for a week.
Thursday (today!): Mini Trypsin Digestion – Jose and Yaamini (ft. Kaitlyn) Friday: I speed vacuum my samples. This way, I can use the weekend to study for my QSCI final and wrangle with Skyline and MSstats. Monday: Desalt all my samples. Forget about all labwork until mid-July. Spend countless hours at Manchester and at the Windows machine instead.
Here’s how Jose and I digested 100 µg of protein from each sample:
Step 1: Set up heating blocks
- Desired temperature is 37 ºC
- Use additional thermometers to confirm temperature
Step 2: Label new tubes for digestion
Step 3: Make 50 mM NH4HCO3 + 6M urea solution
Step 4: Pipet volume of sonicated sample into new tubes
- Based on yesterday’s BCA Assay, I calculated the volume of sonicated smaple necessary for 100 µg of protein
Step 5: Pipet volume of 50 mM NH4HCO3 + 6M urea solution into new tubes
- Combined, the sonicated sample and 50 mM NH4HCO3 + 6M urea solution volume in the new tubes should be 100 µL
Step 6: TCEP Incubation
- Add 6.6 µL of 1.5 M Tris pH 8.8 to each sample
- Add 2.2 200 mM TCEP to each sample
- Vortex gently
- Check pH of solution to ensure it’s still basic
- Pipet 2 µL of solution onto the green square of a pH strip. If the color is blue, it’s basic.
- Place samples on heating block
- Incubate for one hour at 37 ºC
Step 7: IAA Incubation
- Obtain IAA from the freezer. Cover with aluminum foil.
- Add 200 µL IAA to each sample
- Vortex gently
- Cover samples with aluminum foil
- Incubate for one hour at room temperature in the dark
Step 8: DTT Incubation
- Add 200 µL DTT to each sample
- Vortex gently
- Incubate for one hour at room temperature
Step 9: Lys-C Incubation
- Add 1.65 µL Lys-C to each sample
- Vortex genly
- Incubate for one hour at room temperature
Step 10: 25 mM NH4HCO3 and Methanol Addition
- Prepare 25 mM NH4HCO3
- Need 800 µL for each sample
- 100*800 = 80000 µL = 80 mL 25 mM NH4HCO3 needed
- Add 800 µL NH4HCO3 to each sample
- Obtain HPLC grade methanol from flammable storage
- Pour out necessary methanol into a clean beaker
- Need 200 µL for each sample
- 100*200 = 20000 µL = 20 mL methanol needed
- Place methanol in fume hood
- In the fume food, add 200 µL methanol to each sample
- Vortex samples gently
Step 11: Trypsin Digestion
- Obtain 20 µg Trypsin bottles from fridge
- When ready to use Trypsin, add 20 µL nanopure water to the bottle
- Creates a 1 µg/µL solution
- Add 3.3 µL trypsin to each sample
- One bottle provides for 6 samples
- Need about 15 bottles for all samples
- Vortex samples gently
- Let samples sit overnight
- Digestion start:
Step 12: Stop Digestion
- Place all samples in the -80ºC freezer
- Digestion end:
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