goat anti-mouse Immunoglobulins/HRP (Dako, P0447, lot 20058518)
Purify Bb fragment from plasma samples.
Separate 10ng purified Bb fragment and 0.5µl of zymosan activated serum and 0,5 µl of control serums on a 10% SDS-PAGE electrophoresis gel for 1h 120 V.
Activate PVDF membrane with methanol for 15sec and rinse with transfer buffer for 5min. Transfer proteins from the gel to the PVDF membrane using a Mini Trans-Blot cell Tetra Blotting Module (Bio-Rad, 1660827EDU) for 1h at 100V.
Stain membrane using Ponceau S to verify protein transfer.
Block the membrane with blocking buffer (1x PBS, 3% BSA, 0.1% Tween) for 30min at RT.
Incubate membrane with primary mouse anti-complement factor Bb antibody (antibodies-online, ABIN5692886, lot 146096) diluted 1:1000 in working buffer (1x PBS 1% BSA, 0.1% Tween) ON at 4°C.
Wash membrane 3x for 5min with PBST (1x PBS, 0.1% Tween).
Incubate membrane with secondary goat anti-mouse Immunoglobulins/HRP (Dako, P0447, lot 20058518) diluted 1:1000 in working buffer for 30min at RT.
Wash membrane 3x for 5min with PBST.
Reveal protein bands using ClarityTM Western ECL substrate (Bio-Rad, 170-5061, lot 102031078) on a ChemiDoc MP Imaging System (Bio-Rad, 17001402).
In dentatured samples the complement Factor Bb antibody directed against a neo-epitope in Bb fragment ABIN2473066 reveals two proteins of the expected molecular weight of Factor B (90kDa) and Bb fragment (60kDa). The antibody does not specifically recognize a neo-epitope in Bb fragment and is thus not suitable for selectively detecting the Bb fragment subsequently to denaturation.
As an additional control we performed a Western Blot using in-house antibodies which detect Factor B and fragments Bb and Ba (not shown).