Alkaline phosphatase conjugated secondary antibody

Protein concentrations were determined with bicinchoninic acid (BCA) assay (Sigma-Aldrich, St. Louis, MO). SDS-PAGE was performed according to Laemmli [17 (link)]. Separated proteins were transferred to nitrocellulose membranes (Millipore, MA). Blots were probed with primary antibodies, followed by an alkaline phosphatase-conjugated secondary antibody (12500-fold dilution, DAKO, Glostrup, Denmark). The reaction was developed using a BCIP/NBT phosphatase system (KPL, Gaithersburg, MD) as described previously [18 (link)–20 (link)]. Optic pathways from knockout and wild-type controls were homogenized in 250μl μl of buffer (SDS buffer) containing 25 mM sodium phosphate (pH 7.2), 5 mM EGTA, 1% SDS, and 1 mM phenylmethylsulfonyl fluoride and protein concentration was measured with the BCA assay (Sigma-Aldrich, St. Louis, MO). Loading onto gels was normalized to total protein of the optic axons.

The 14H6 chimeras were analyzed by SDS-PAGE using methods outlined by Laemmli with slight modifications. Protein samples were mixed with equal volumes of 6× loading buffer (composed of 100 mM Tris-HCl, pH 6.8, 200 mM BME, 4% SDS, 0.2% Bromophenol blue and 20% glycerol). Sample mixtures were heated at 80°C for 10 min and subsequently loaded onto 10% acrylamide gels. For western blotting analyses, separated samples of the 14H6 chimeras were transferred from the SDS gels to nitrocellulose membranes. The membranes were blocked with 5% skim milk, soaked in 1:500 diluted HPV16 L2-specific mice sera, and incubated at room temperature for 1 h. Subsequent washing was performed using 0.2% Tween-20 in phosphate-buffered saline, pH 7.4. Alkaline phosphatase-conjugated secondary antibody (Dako, Denmark) was then added to capture the bound primary antibody. A mixture of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate was then added to allow color development and visualization of the target protein bands.