Amersham hybond p0.2 pvdf

For pull-down assay, 0.1 mg/mL of purified protein was immobilized on Pierce NHS-activated magnetic beads (ThermoFisher) following the manufacturers’ instructions. The beads were incubated with 10 mM EDTA and 50 μL serum (21 °C, 30 min). The beads were washed 3 times with 1 mL PBS + 0.05% Tween20, once with 100 μL PBS, and boiled in 50 μL SDS- PAGE loading buffer. The eluted proteins were separated on an SDS polyacrylamide gel and observed by Coomassie staining, silver staining, or Western blotting. For blotting the SDS/PAGE-separated proteins were transferred to a PVDF membrane (Amersham Hybond P0.2 PVDF, 55 GE) by semiwet transfer (Bio-Rad) and blocked for 1 h with 2% milk. Primary antibody (α-C5: 1:80,000, Complement Technology). Secondary antibody (α-goat HRP, Promega, 1:10,000). The blot was developed using ECL Western Blotting Substrate (Promega) and imaged using Amersham Hyperfilm ECL (GE Healthcare).

Samples were separated on 4–12% Bis–Tris polyacrylamide gel (Invitrogen) followed by transfer to Amersham™ Hybond® P 0.2 PVDF (GE Healthcare) for immunoblotting. Primary antibodies: anti- anti-ELP1 (1:250, Abcam, ab56362), anti-ELP3 (1:1000, Abcam, ab96781), anti-AAG (1:1000, custom rabbit polyclonal antibody, Covance, raised against ∆80AAG) or anti-AAG (1:1000, LSBio LS-C133325), anti-RNA pol II S2P (1:1000, Abcam, ab5095), anti-HA (1:1000, Abcam, ab9110), anti-α-Tubulin (1:10000, Cell Signaling, 2144), anti-H3 (1:1000, Abcam, ab1791), anti-GFP (1:1000, Abcam, ab290); were detected using infrared (IR) Dye-conjugated secondary antibodies (1:15000, Li-COR Bioscienecs, 827-11081 and 925-32210). The signal was visualized by using direct IR fluorescence via the Odyssey Scanner, LI-COR Biosciences.

For blotting the SDS/PAGE-separated proteins were transferred to a PVDF membrane (Amersham Hybond P0.2 PVDF, 55 GE) by semiwet transfer (Bio-Rad) and blocked for 1 h with PBS/2% milk.
Primary antibodies were purchased from CompTech (Complement Technologies Inc, USA): goat α-properdin, 1:2,000, goat α-Factor B, 1:4000, goat α-Factor D, 1:250. Secondary antibody (donkey α-goat HRP, Promega, 1:10,000). For His-tagged proteins, the Penta-His HRP Conjugate Kit (Qiagen) was used following the manufacturer’s instructions.
Blots were developed using ECL Western Blotting Substrate (Promega) and imaged using Amersham Hyperfilm ECL (GE Healthcare) or using a ChemiDoc XRS + imaging system (Biorad).