Subsequent to this successful outcome, another laboratory (RHA) was attempting to identify peptides secreted by cultured choroid plexus epithelium. This required analysis of the tissue culture medium. Although the serum levels in the medium could be lowered from 10% to 2% for short periods, this condition compromised the cells over a longer period of time. Further, these "low" serum levels still have massive amounts of albumin, overwhelming any HPLC separations attempted.
However, if the culture medium was passed over a column of Blue-Sepharose (Sigma), HPLC separations with significant detail were obtained. Specifically, transthyretin, a 12,000 Dalton protein which binds thyroxin and retinoic acid, known to be secreted only by choroid plexus and liver, was clearly identified. Although this protein was eluted from a C-18 column slightly before the peak of remaining bovine albumin, unequivocal sequence data were obtained, free from any contaminating albumin sequences.
The Blue-Sepharose gels can be purchased either as loose media or as small, prepacked columns from companies such as Sigma or Pharmacia-LKB, while Affi-Gel Blue can be purchased from BioRad. The companies provide protocols for use.1 The resins will specifically retain enzymes which have binding sites for adenylate cofactors, in addition to binding serum albumin. Some proteins may also bind by hydrophobic interactions. Generally, the samples are loaded at pH 7.5-8.0 and low salt concentrations. If elution of the bound proteins is desired, then 1.5 M NaCl in buffer, chaotropic agents, or more specific eluants can be used. In the above examples, where the primary need is to remove the albumin, there is no need to elute unless the resin will be reused. The breakthrough of the column is the desired sample. We have found we can either load tissue culture media directly, at physiological salt concentrations, or after diluting it 1:1 in distilled water. For the first application above, a 3 ml column is sufficient. Small fractions were collected, and the samples were run on SDS-PAGE without further concentration. In the second application, 10 ml columns packed in the laboratory were used, one for the control and one for the experimental medium. When one ml of the breakthrough was injected directly on a 2.1 mm x 23 cm C-18 column using a HP1090 HPLC, a transthyretin peak of more than 0.125 absorbance at 214 nm was obtained.
1 Additional information can be found in Hermanson, G.T., Mallia, A.K., & Smith, P.K. (1992) Immobilized Affinity Ligand Techniques, Academic Press, pp 454.
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