Difference in CYP2D6 immunoblot linear calibration range and holoprotein/total CYP2D6 protein ratio between CYP2D6 Supersomes and in-house purified CYP2D6 allelic isoforms
Values represent mean of duplicate or triplicate measurements. Total protein concentration was determined using the BCA Protein Assay, and CYP2D6 holoprotein content was determined according to the CO difference spectrum. Total CYP2D6 concentration for in-house purified CYP2D6 isozyme was calculated according to its average molecular mass.
Sample | Molecular Mass | Total Protein | CYP2D6 Holoprotein | Total CYP2D6 | Holoprotein/Total CYP2D6 Protein | Immunoblot Linear Range |
|---|---|---|---|---|---|---|
| Da | mg/ml | pmol/μl | pmol/μl | % | pmol | |
| CYP2D6.1 Supersomes | 55,769.5 | 3.20 | 1.00 | 2.82a | 35.5a | 0.025-0.25 |
| CYP2D6.10 Supersomes | 55,773.4 | 1.10 | 1.00 | 31.6a | 3.16a | 0.025-0.25 |
| CYP2D6.1 (in-house) | 55,769.5 | 0.60 | 6.90 | 9.72b | 71.0b | 0.05-0.50 (Fig. 1); 0.10-1.0 (Fig. 2) |
| CYP2D6.2 (in-house) | 55,730.4 | 0.60 | 5.00 | 9.72b | 51.4b | 0.10-1.0 |
| CYP2D6.10 (in-house) | 55,773.4 | 11.3 | 1.80 | 142b,c | 1.27b,c | 0.01-0.10 |
↵ a Estimated after compared with corresponding in-house CYP2D6 allelic isoforms (Fig. 1).
↵ b Values corrected with the purity of CYP2D6.
↵ c Low level of CYP2D6.10 holoprotein as a result of enzyme denaturation during purification (Yu et al., 2002).