Immunohistochemical staining of paraffin embedded human endometrium with purified ab109185 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. 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.

All lanes : Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : MUC1 knockout HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 17 kDa
Observed band size: 17-24 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab109185 observed at 24 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab109185 was shown to specifically react with MUC1 in wild-type HeLa cells as signal was lost in MUC1 knockout cells. Wild-type and MUC1 knockout samples were subjected to SDS-PAGE. Ab109185 and ab9484 (Mouse 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-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.

Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution (Purified antibody) + Colon cancer at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 17 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded mouse lung with purified ab109185 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. 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.

Immunohistochemical staining of paraffin embedded rat kidney with purified ab109185 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. 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.

Immunofluorescence staining of A431 cells with purified ab109185 at a working dilution of 1 in 1000, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 555 goat anti rabbit, used at a dilution of 1 in 400. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab109185 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500.

Anti-MUC1 antibody [EPR1023] (ab109185) at 1/5000 dilution (Purified) + T47D cell lysate at 10 µg

Secondary
Secondary ab: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 17 kDa
Observed band size: 18-25 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab109185 (unpurified) showing positive staining in Breast ductal infiltrating carcinoma tissue.

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

ab109185 (unpurified) showing positive staining in Normal stomach tissue.

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

ab109185 (purified) at 1/20 immunoprecipitating MUC1 in T47-D (Lane 1). Lane 2 - 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.

Diluting buffer and concentration: 5% NFDM /TBST.

Overlay histogram showing MCF7 cells fixed in 2% PFA and stained with purified ab109185 at a dilution of 1 in 30 (pink line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control.

All lanes : Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution (Unpurified)

Lane 1 : T47-D cell lysate
Lane 2 : Human colon cancer lysate

Lysates/proteins at 10 µg per lane.

Performed under reducing conditions.

Predicted band size: 17 kDa
Additional bands at: 17 kDa (possible cleavage fragment)

Overlay histogram showing MCF7 cells stained with unpurified ab109185 (red line). The cells were fixed with 80% 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 (ab109185, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Equilibrium disassociation constant (K_D) of unpurified ab109185.
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