Antibody specificity and protein linearity
Pooled Control group lung membranes from the lower and upper lung were
serially diluted to generate a standard curve to assess the linear
detection range of CYP2A, β-actin and total proteins (i.e. Coomassie
blue-stained protein). Individual AGM samples were loaded at 75μg beside
the pooled Control standard curve at 25μg, 75μg, and 150μg on each
immunoblot. The standard curve was used to ensure that each membrane
sample was in the linear range and to allow for blot-to-blot
adjustments. Each blot had a pre-stained protein mixture in one lane for
molecular weight estimation (PaperRuler Prestained Protein Ladder,
ThermoFisher; Mississauga, ON, CA).
The specificity and sensitivity of the polyclonal anti-CYP2A6 antibody
was assessed using positive controls including human liver microsomes,
AGM liver microsomes, AGM lung membranes and membranes from cDNA
expressed human CYP2A6 (Corning; Corning NY, USA), and CYP2A13
(XenoTech; Lenexa, KS, USA). Negative controls included cDNA expressed
human CYP2E1 (BD Bioscience; Woburn, MA, USA), CYP2D6 (XenoTech; Lenexa,
KS, USA), CYP2B6 (BD Bioscience; Woburn, MA, USA), and CYP3A4 (BD
Bioscience; Woburn, MA, USA).