Lidstrom:Hot water extraction in vitro
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Day 1: Beginning with frozen lysate samples (150 uL)
- 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
- Boil filtered sterile water in the microwave (3 min).
- Put boiling water in boiling water bath to keep hot while doing the next step
- Add 0.45 (3x) mL boiling water to each sample, cap tightly and vortex to mix.
- Put tubes in boiling water for 1 min. Make sure the water level in the bath is above the liquid level in the tubes.
- Put the tubes on ice for 10 min. Use a deep ice bucket.
- Pre-cool centrifuge.
- Vortex tubes for 1 min each.
- Centrifuge 5 min, 14,000rpm, 4°C
- Decant into fresh tubes.
- 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.
- Flash freeze clean supernatants with liquid Nitrogen.
- Pause Point: Samples can be stored at -80°C.
- Open tubes and cover with parafilm. Puncture holes in parafilm. Put tubes in foam holder
- Lyophilize tubes for until dry (~overnight)
Day 2: Reconstitute #2
- Reconstitute in 100 uL sterile, filtered ddH2O.
- Centrifuge tubes, max speed, 4°C, for x 10 minutes
- Filter supernatant with spin column filter (centrifuge until all liquid has passed through the filter)
- Pipet sample into vial insert in MS vials with split lids
- Sample is ready for LC-injection!