All lanes : Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/500 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : DUSP6 knockout A431 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 42 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab76310 observed at 42 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab76310 was shown to recognize in wild-type A431 cells as signal was lost at the expected MW in DUSP6 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and DUSP6 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab76310 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical staining of paraffin embedded human pancreas with purified ab76310 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunoblot analysis of indicated proteins in splenocytes after CD4+ sort, liver homogenate or purified CD4+ T cells from WT, DUSP6+/- or DUSP6-/- mice to confirm knockout or heterozygosity for DUSP6 at protein level.

This image was generated using ab220811, the same antibody but in a PBS-only buffer formulation. 

Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/5000 dilution (purified) + HepG2 cell lysate at 20 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 42 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

ab76310 detects an unspecific band around 100 kDa in human materials.

Overlay histogram showing NIH-3T3 cells fixed in 4% PFA and stained with purified ab76310 at a dilution of 1 in 200 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

ab76310 (purified) at 1/20 immunoprecipitating DUSP6 in 10 μg NIH-3T3 (Lanes 1 and 2, observed at 42 and 44 kDa). Lane 3 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

All lanes : Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/1000 dilution (purified)

Lane 1 : rat brain lysate
Lane 2 : NIH/3T3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/1000 dilution (purified) + mouse brain at 10 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 42 kDa
Observed band size: 42,44 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/500 dilution (unpurified) + 3T3 cell lysate at 10 µg

Secondary
HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 42 kDa
Observed band size: 42/44 kDa why is the actual band size different from the predicted?

All lanes : Anti-DUSP6 antibody [EPR129Y] (ab76310) at 1/4000 dilution (unpurified)

Lane 1 : Marker
Lane 2 : Lysate from wild type primary murine embryonic fibroblasts (MEFs) untreated at 10 µg
Lane 3 : Lysate from wild type primary murine embryonic fibroblasts (MEFs) treated with 100ngµl-1, 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 hours at 10 µg
Lane 4 : Lysate from MKP-3 null primary murine embryonic fibroblasts (MEFs) untreated at 10 µg
Lane 5 : Lysate from MKP-3 null primary murine embryonic fibroblasts (MEFs) treated with 100ngµl-1, 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 hours at 10 µg

Secondary
All lanes : Goat anti Rabbit HRP conjugate at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 2 minutes

See Abreview

Immunohistochemical staining of paraffin-embedded human gastric carcinoma using unpurified ab76310 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with unpurified ab76310 (red line). The cells were fixed with methanol (5 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 (ab76310, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized with 0.1% PBS-Tween 20 used under the same conditions.