All lanes : Anti-pan Cadherin antibody (ab16505) at 1 µg/ml

Lane 1 : Human heart lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat heart lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab7090) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 100 kDa
Observed band size: 125-140 kDa why is the actual band size different from the predicted?
Additional bands at: 40 kDa, 65 kDa, 75 kDa, 90 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

ICC/IF image of ab16505 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16505, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

IHC image of pan Cadherin staining in human liver FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16505, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

All lanes : Anti-pan Cadherin antibody (ab16505) at 1 µg/ml

Lane 1 : Mouse heart
Lane 2 : HeLa cell lysate
Lane 3 : 3T3 cell lysate
Lane 4 : Mouse muscle
Lane 5 : Human heart
Lane 6 : Mouse heart with Human pan Cadherin peptide (ab17098) at 1 µg/ml
Lane 7 : HeLa cell lysate with Human pan Cadherin peptide (ab17098) at 1 µg/ml
Lane 8 : 3T3 cell lysate with Human pan Cadherin peptide (ab17098) at 1 µg/ml
Lane 9 : Mouse muscle with Human pan Cadherin peptide (ab17098) at 1 µg/ml
Lane 10 : Human heart with Human pan Cadherin peptide (ab17098) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-rabbit conjugated to Alexafluor 680 at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 100 kDa

HeLa cells fixed in methanol and stained with ab16505 (2µg/ml). The cells were fixed in 100% methanol for 6 minutes at -20°C, then washed once in PBS. The 2 images show the cells stained with different secondary antibodies, Donkey anti Rabbit FITC (image A) and Donkey anti Rabbit Cy3 (image B). In each case ab16505 stains the plasma membrane.  In image A ab16505 is stained green and in image B ab16505 is stained red. In both images the DNA is stained with DAPI (blue).

Immunofluorescence analysis of HeLa cells, staining pan Cadherin (red) with ab16505.

Cells were fixed with paraformaldehyde, permeabilized in methanol and blocked for 1 hour at room temperature in DPBS containing 2 mg/mL BSA, 1% fish gelatin, 0.1% Triton-X 100 and 5% goat serum. Cells were then incubated for 1 hour at room temperature with the primary antibody diluted in blocking buffer. An AlexaFluor®-conjugated anti-rabbit IgG was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).