Atlas Biological does not perform charcoal treatments to serums orders. Directions are provided for clients to perform this procedure in-house.
The following protocol is suggested for the removal from most of the hormones from serum.
Charcoal treated, charcoal filtered or charcoal stripped bovine serum — exposed serum to a mixture of dextran coated activated charcoal for a period of time.
- Vortex or bench mixer
- Fetal Bovine Serum or Bovine Serum
- Norit-A charcoal
- Dextran T-70
- 1.5mM MgCl2
- 10mM HEPES
- Phosphate Buffered Saline
- Demonized Water – low endotoxin
- Sterile conical tubes
- 0.45 micron filter
- 0.2 micron filter
Charcoal treatment of serum is an effective process for removing hormones and steroids. It has also been used for improving immunoassay systems. Several methods are utilized, material and concentrations are similar.
For laboratory use only
- Preparation of Dextran-coated charcoal.
- Incubate overnight at 4°C 0.25% Norit A charcoal and 0.0025% Dextran T-70 in 0.25M sucrose, 1.5mM MgCl2 10mM HEPES, pH 7.4.
- Remove volume of dextran-coated charcoal equivalent to the serum volume required. Centrifuge 500xg for 10 minutes to pellet charcoal.
- Removed and discard supernatant and suspend with low endotoxin de-ionized water to wash the pellet.
- Centrifuge 500xg for 10 minutes to pellet charcoal.
- Remove and discard supernatant and suspend with equal volume of bovine serum for an equivalent of 253 mg of dextran-coated charcoal per 100mL of serum.
- Vortex to mix and incubate for 12 hours at 4°C or 2 times for 45 minutes at 56°C.
- Pass the stripped serum through a pre-filter, 0.45 micron and 0.2 micron filter prior to application. Store at -10 to -30°C.