All lanes : Anti-Desmoglein 3/PVA antibody [5H10] (ab231309) at 5 µg/ml

Lane 1 : A431 Whole Cell Lysate
Lane 2 : Human Skin Tissue Lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 108 kDa

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 blocked for an hour using 3% milk before ab231309 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 5ug/ml concentration and 1/20000 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.

IHC image of ab231309 staining in 10% formaldehyde fixed frozen tissue section of normal human skin.

Non-specific protein-protein interactions were blocked using TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1 hour at room temperature. The section was then incubated with ab231309 (1 μg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. The section was then incubated with ab150119 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647)) and DAPI for 1 hour at room temperature.

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is derived directly from bound ab231309. 

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such antibody concentrations and incubation times.

ab231309 staining Desmoglein 3/PVA in A431 (human epidermoid carcinoma cell line) cells.

The cells were fixed with 100% methanol (5 minutes), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated overnight at +4°C with ab231309 at 1 μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red).

Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).