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 ab231078 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.

All lanes : Anti-C3b / iC3b antibody [7C12] (ab231078) at 1 µg/ml

Lane 1 : Human Sera diluted 1/1000 at 10 µl
Lane 2 : Purified C3 Protein at 0.1 µg
Lane 3 : Purified C3b Protein at 0.1 µg
Lane 4 : Purified iC3b Protein at 0.1 µg

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 110,125,43,78 kDa why is the actual band size different from the predicted?

This blot was produced using a 4-12% Bis-tris 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 ab231078 overnight at 4°C at 1ug/ml. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes :

Lane 1 : Human serum diluted 1/100 at 5 µl
Lane 2 : Human plasma diluted 1/100 at 5 µl
Lane 3 : Human liver whole tissue lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 68 kDa
Additional bands at: 42 kDa (possible cleavage fragment)

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 ab231078 and ab181602 (Rabbit anti GAPDH) were incubated overnight at 4°C at a 1ug/ml concentration and 1/10000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.