- Naphthalene treatment: i.p. application of 200mg per kg body weight (KG) naphthalene dissolved in oil.
- Denature 100µg total protein for 5min at 95°C in Laemmli SDS sample buffer and subsequently separate them on a denaturing gel SDS-PAGE gel in for 90min at 100V.
- Transfer proteins onto 0.2µm nitrocellulose membrane in transfer buffer (15% MeOH, Tris 25mM, 86mM, SDS 70mM).
- Block the membrane with blocking solution (PBS, 5% milk).
- Incubation with primary
- rabbit anti-cytochrome P450, family 4, subfamily B, polypeptide 1 (CYP4B1) (N-Term) antibody (antibodies-online, ABIN2777006, lot QC12731-90422) diluted 1:300 or
- Hsc70 loading control antibody (supplier, product no, lot no) diluted
- in blocking solution ON at 4°C.
- Wash membrane 3x with blocking solution.
- Incubate with secondary donkey anti-rabbit IgG (H+L) peroxidase-conjugated F(ab')2 fragment (Jackson ImmunoResearch) diluted1:10000 in blocking solution for 1h at RT.
- Wash membrane 3x with PBS, 1x with blocking solution, 2x with PBS.
- Develop membrane with SuperSignal West femto substrate (Thermo Fisher Scientific, 34095) according to manufacturer’s recommendations, exposure time 25min.
- Repeat the incubation steps with both antibodies, washes, and revealing the protein bands.