Charcoal Treated Bovine Serum Protocol

Purpose

This protocol is suggested for the removal of most hormones and steroids from serum using dextran-coated activated charcoal.

Definition

Charcoal treated, charcoal filtered, or charcoal stripped serum: Serum exposed to a mixture of dextran-coated activated charcoal for a specified period of time.

Equipment

• Centrifuge
• Vortex or bench mixer

Materials

• Fetal Bovine Serum or Bovine Serum
• Norit-A charcoal
• Dextran T-70
• 1.5mM MgCl2
• 10mM HEPES
• Phosphate Buffered Saline
• Deionized Water – low endotoxin
• Sterile conical tubes
• 0.45 micron filter
• 0.2 micron filter

Specifications

Charcoal treatment is effective for removing hormones and steroids and can improve immunoassay performance. Methods may vary slightly, but materials and concentrations are generally similar.

Precautions

Procedure

1. Preparation of Dextran-Coated Charcoal: Incubate overnight at 4°C with 0.25% Norit-A charcoal and 0.0025% Dextran T-70 in 0.25M sucrose, 1.5mM MgCl2, 10mM HEPES, pH 7.4.
2. Remove volume of dextran-coated charcoal equivalent to the required serum volume. Centrifuge at 500 x g for 10 minutes to pellet charcoal.
3. Discard supernatant and resuspend pellet with low endotoxin deionized water to wash. Centrifuge again at 500 x g for 10 minutes.
4. Discard supernatant and resuspend charcoal with an equal volume of serum, using ~253 mg of dextran-coated charcoal per 100 mL of serum.
5. Vortex to mix thoroughly.
6. Incubate for 12 hours at 4°C or 2 cycles of 45 minutes at 56°C.
7. Filter the treated serum through a pre-filter, then 0.45 micron and 0.2 micron filters before use.
8. Store treated serum at -10 to -30°C until application.

Charcoal Treatment Workflow