genefish 05:37 on June 1  

Yaamini’s Notebook: DNR Mini Trypsin Digestion Round 3

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Six boxes of trypsin arrived yesterday. Guess what that means?!

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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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