Mab4165

Recombinant human type 23 collagen (R&D system, 4165-CL) was diluted in sample buffer containing 80 mM dithiothreitol (DTT) and run on a 10% SDS-PAGE gel, and subsequently transferred onto a nitrocellulose membrane. The nitrocellulose membranes were then blocked for non-specific binding by incubation for 1 hour at room temperature in tris-buffered saline-Tween® 20 (TBS-T) buffer containing 5% skim milk powder. This was followed by incubation with 1 µg/ml 10F6 or commercial type 23 collagen antibody (R&D system, MAB4165) diluted in TBS-T milk for overnight.
The recombinant type 23 collagen for both commercial Ab and 10F6 were prepared together. Half volume of recombinant protein was loaded for commercial Ab incubation. The other half volume was loaded for 10F6 incubation. The loading and transfer were done using the same gel, which ensures the equal transfer time. The recombinant protein demonstrated a 95% purity, which made it unnecessary to normalize total protein expression Then the membranes were washed in TBS-T 3 times, followed by incubation in the secondary peroxidase-conjugated antibody. The secondary Ab (Jackson, 315-035-045, 1:5000 dilution) was incubated at room temperature for 1 hour. Finally, the membranes were washed in TBS-T 3 times, and the results were visualized using the enhanced chemiluminescence (ECL) system (GE healthcare, cat# RPN2109).