All lanes : Anti-CD105 antibody [MEM-226] (ab2529) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : CD105 knockout HeLa whole cell lysate
Lane 3 : HUVEC whole cell lysate

Lysates/proteins at 20 µg per lane.

Lanes 1 - 3: Merged signal (red and green). Green - ab2529 observed at 70 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab2529 was shown to recognize ENG (Endoglin) in wild-type HeLa cells as signal was lost at the expected MW in ENG knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and ENG knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab2529 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing U937 (Human histiocytic lymphoma cell line) cells stained with ab2529 (red line).

The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2529, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in U937 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ICC/IF image of ab2529 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were 4% formaldehyde fixed (10 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2529, 10 µg/ml) overnight at +4°C. The secondary antibody (green) was ab69879, DyLight^® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

Standard Curve for CD105 (Analyte: CD105 protein (ab54338)); dilution range 1pg/ml to 1ug/ml using Capture Antibody Mouse monoclonal [MEM-226] to CD105 (ab2529) at 5ug/ml and Detector Antibody Rabbit polyclonal to CD105 (ab21224) at 0.5ug/ml.

Anti-CD105 antibody [MEM-226] (ab2529) at 5 µg/ml + Human colon tissue lysate at 10 µg

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Observed band size: 80 kDa why is the actual band size different from the predicted?
Additional bands at: 45 kDa, 55 kDa. We are unsure as to the identity of these extra bands.