Anti-C3b / iC3b antibody [1H8]

• ELISA

Lab

ELISA - Anti-C3b / iC3b antibody [1H8] (AB231080)

96-well microtitre plates were coated overnight at 4°C with recombinant human C3, C3b, iC3b, and Factor H proteins, in duplicate at a concentration of 1μg/mL. Plates were blocked with 1% BSA in PBS-T (0.1% Tween®) for 1 hour before incubation with a 10-step 4x serial dilution of ab231080 from 10μg/mL for 1 hour at room temperature. Antibody binding was detected with Goat Anti-Mouse IgG H&L (HRP) (ab6789) secondary antibody at a 1 in 10000 dilution for 1 hour at room temperature. Plates were incubated with TMB ELISA substrate for 7 minutes prior to being stopped with Stop solution and absorbance measured at 450nm.

• WB

Lab

Western blot - Anti-C3b / iC3b antibody [1H8] (AB231080)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% milk before being incubated with ab231080 overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-C3b / iC3b antibody [1H8] (ab231080)

Lane 1:

human serum diluted 1/100 at 5 µL

Lane 2:

Human plasma diluted 1/100 at 5 µL

Predicted band size: 187 kDa

Observed band size: 68 kDa

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