Lidstrom:Hot water extraction in vitro

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Day 1: Beginning with frozen lysate samples (150 uL)

1. Preheat hot water baths to 100°C (~1 hr before using). Add additional DI water so it will cover the most of the tubes. °C
2. Boil filtered sterile water in the microwave (3 min).
3. Put boiling water in boiling water bath to keep hot while doing the next step
4. Add 0.45 (3x) mL boiling water to each sample, cap tightly and vortex to mix.
5. Put tubes in boiling water for 1 min. Make sure the water level in the bath is above the liquid level in the tubes.
6. Put the tubes on ice for 10 min. Use a deep ice bucket.
7. Pre-cool centrifuge.
8. Vortex tubes for 1 min each.
9. Centrifuge 5 min, 14,000rpm, 4°C
10. Decant into fresh tubes.
11. The remaining tubes containing protein and cell debris were centrifuged again, at 4°C, 14,000rpm for 5 minutes, and supernatants were removed carefully into the clean tubes.
12. Flash freeze clean supernatants with liquid Nitrogen.

• Pause Point: Samples can be stored at -80°C.

1. Open tubes and cover with parafilm. Puncture holes in parafilm. Put tubes in foam holder
2. Lyophilize tubes for until dry (~overnight)

Day 2: Reconstitute #2

1. Reconstitute in 100 uL sterile, filtered ddH2O.
2. Centrifuge tubes, max speed, 4°C, for x 10 minutes
3. Filter supernatant with spin column filter (centrifuge until all liquid has passed through the filter)
   • [|Costar 8161 Spin-X Centrifuge Tube Filter, 0.22μm cellulose acetate in a 2.0 mL polypropylene tube. non-sterile, 100/case]
4. Pipet sample into vial insert in MS vials with split lids
5. Sample is ready for LC-injection!