A4062

Polyacrylamide gels were equilibrated in transfer buffer (48 mM Tris, 39 mM glycine, 1.3 mM SDS, 20% methanol) for 20 min. A polyvinylidene difluoride (PVDF) membrane was activated in 100% methanol for 1 min. Filter paper and activated PVDF membranes were saturated in transfer buffer and proteins transferred using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was washed in TBST (10 mM Tris-Cl, 200 mM NaCl, 0.1% Tween 20, pH 7.5), blocked in 5% bovine serum albumin, and probed overnight at 4° using 1:2000 mouse anti-H3K9me2 diluted in blocking solution (ab1220; Abcam) or 1:4000 goat anti-tubulin (E7; Developmental Studies Hydrinoma Bank). After washing with TBST, the membrane was incubated with alkaline-phosphatase-conjugated secondary antibodies (A3562, goat anti-mouse; Sigma, or A4062, rabbit anti-goat; Sigma), washed and developed in 100 mM diethanolamine, 100 mM NaCl, 5 mM MgCl₂, pH 9.5 containing 33 µg/ml nitroblue tetrazolium (NBT) and 165 µg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Signals were quantified by ImageJ.

Murine monoclonal anti-human C1q (#A201, Quide San Diego, CA) (25 μl/well of 2μg/ml) in PBS was dry-coated into 96-well polystyrene microtiter plates (Costar, 3690, Corning) overnight at 4 °C. Wells were blocked 3% non-fat dry milk with 50ul/well (# M0841, LabScientific Highlands, NJ) in PBS for 4 h at room temperature. After rinsing the wells three times with PBS-0.05%Tween, 25 μl of serum samples diluted in PBS were added to each well. The serum dilutions were obtained by first making a 1:100 dilution and serially re-diluting this solution until it was 1:10,000. Samples were incubated overnight at 4 °C. Wells were then washed 3 times with PBS- Tween. Goat antiserum to human C1q (#A301, Quidel) was diluted 1:1000 in 0.3% non-fat milk in PBS and added (25 μl/well) for 2 h at room temperature. After washing 3 times in PBS- Tween, plates were incubated for 1 h at room temperature with rabbit anti-goat IgG antibody conjugated to alkaline phosphatase (#A-4062, Sigma) diluted in 0.3% non-fat milk in PBS at 1:500. The wells were washed 3 times with PBS-Tween and incubated with 50 μl of alkaline phosphatase substrate (Sigma) in solution (.5 M Na2CO3 and .01 M MgCl2) (check). The absorbance of each well was read at 30 min at 405 nm. The standard curve of purified human C1q was linear in the 2 ng to 250 ng range. Both the standards and serum samples were assayed in triplicate.