Anti-EDC4 antibody (ab72408)

  Western blot - EDC4 antibody (ab72408)

All lanes : Anti-EDC4 antibody (ab72408) at 0.04 µg/ml

Lane 1 : Whole cell lysate from Hela cells at 50 µg
Lane 2 : Whole cell lysate from Hela cells at 15 µg
Lane 3 : Whole cell lysate from Hela cells at 5 µg
Lane 4 : Whole cell lysate from 293T cells at 50 µg
Lane 5 : Whole cell lysate from NIH3T3 cells at 50 µg

developed using the ECL technique

Predicted band size : 152 kDa
Observed band size : 190 kDa (why is the actual band size different from the predicted?)
Additional bands at : 55 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 30 seconds

  Immunoprecipitation - EDC4 antibody (ab72408)

Immunoprecipitation/ Western Blot of EDC4.
Lane 1: ab72408 at 3µg/mg whole cell lysate.
Lane 2: Control IgG.

ab72408 at 1µg/ml for WB.
Whole cell lysate from Hela cells at 1mg for IP, 20% of IP loaded.
Chemiluminescence with an exposure time of 3 seconds.

  Immunocytochemistry/ Immunofluorescence-EDC4 antibody(ab72408)

ICC/IF image of ab72408 stained HeLa 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 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab72408, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - EDC4 antibody (ab72408)

IHC image of ab72408 staining in human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab72408, 5µg/ml, for 15 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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.