Plasma

Collect blood samples into the Lavender Vacutaner tubes which contain EDTA and can collect 7 ml blood/tube. Gently rock the Lavender Vacutaner tubes several times immediately after collection of blood for anti-coagulation. Transfer the blood from the Lavender Vacutaner tubes to centrifuge tubes containing aprotinin (0.6 TIU/ml of blood) and gently rock for several times to inhibit the activity of proteinases. Centrifuge the blood at 1,600 x g for 15 minutes at 4°C and collect the plasma. Plasma kept at -70°C may be stable for one month.

Extraction

Plasma

• Acidify 1 ml of plasma with 0.5 ml 1.5% trifluoroacetic acid (TFA).
• Centrifuge acidified samples at 16,000 x g for 20 minutes; transfer supernatants to fresh tubes.
• Equilibrate Strata X 96 well plate (60 mg resin/well; Phenomenex Cat No. 8E-S100- UGB*) with 1 ml of methanol followed by 1 ml of deionized water.
• Perform under low vacuum.
• Load cleared samples onto the Strata X plate.
• Wash plates with 1.5 ml 0.1% TFA.
• Elute samples with 1 ml acetonitrile/H2O (80:20).
• Dry down pellets under vacuum.
• Resuspend pellets in 0.125 ml β-endorphin assay buffer.
• Extraction (1 ml to 0.125 ml).

Note: An appropriate vacuum manifold (available through Phenomenex) is required to use Strata X96 well plates.

Tissue

Buffers:

• Lysis Buffer: 10mM Tris, pH 7.4
• Buffer A: 1% trifluoracetic acid (TFA, HPLC grade) in H2O
• Buffer B: 60% acetonitrile (HPLC grade) in 1% TFA

Extraction of peptide from tissue homogenates:

1. Remove livers, lungs, kidneys, hearts, and brains and immediately freeze in liquid nitrogen and stored at -80°C.
2. Homogenize tissues in 5 mL/g lysis buffer.
3. Centrifuge for 15 min at 1,600 g at 4°C
4. Load the supernatant on a equilibrated SEP-Pak C18 cartridge (Millipore).
5. Equilibrate the column by washing once with 1 mL of Buffer B and three times with 3 mL of Buffer A.
6. Elute peptide with 3 mL of Buffer B and collect in a 15 mL polystyrol tube, evaporate to dryness, and dissolve the residue in 250 µL of Assay buffer.