Anti-MUC1 antibody [EPR1023]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUC1 antibody [EPR1023] (AB109185)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MUC1 antibody [EPR1023] (AB109185)

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 Fluorr^® 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUC1 antibody [EPR1023] (AB109185)

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

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MUC1 antibody [EPR1023] (AB109185)

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MUC1 antibody [EPR1023] (AB109185)

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.

• IP

Lab

Immunoprecipitation - Anti-MUC1 antibody [EPR1023] (AB109185)

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.

All lanes:

Immunoprecipitation - Anti-MUC1 antibody [EPR1023] (ab109185)

Predicted band size: 122 kDa

Observed band size: 18-25 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUC1 antibody [EPR1023] (AB109185)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUC1 antibody [EPR1023] (AB109185)

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.

• WB

Lab

Western blot - Anti-MUC1 antibody [EPR1023] (AB109185)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MUC1 antibody [EPR1023] (ab109185) at 1/5000 dilution

All lanes:

T47D cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 122 kDa

Observed band size: 18-25 kDa

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• WB

Lab

Western blot - Anti-MUC1 antibody [EPR1023] (AB109185)

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.

All lanes:

Western blot - Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution

Lane 1:

Wild-type HeLa whole cell lysate at 20 µg

Lane 2:

MUC1 knockout HeLa whole cell lysate at 20 µg

Predicted band size: 122 kDa

Observed band size: 17-24 kDa

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• WB

Supplier Data

Western blot - Anti-MUC1 antibody [EPR1023] (AB109185)

Blocking and dilution buffer : 5% NFDM/TBST.

ab181602 was used as loading control at 1/1000000 dilution.

No expression of MUC1 was detected in normal liver (PMID : 16094706).

All lanes:

Western blot - Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution

Lane 1:

Mouse lung tissue lysate at 20 µg

Lane 2:

Rat lung tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Rat liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 122 kDa

Observed band size: 17-24 kDa

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Exposure time: 20s

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUC1 antibody [EPR1023] (AB109185)

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

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

• WB

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Western blot - Anti-MUC1 antibody [EPR1023] (AB109185)

All lanes:

Western blot - Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution

Lane 1:

T47-D cell lysate at 10 µg

Lane 2:

Human colon cancer lysate at 10 µg

Predicted band size: 122 kDa

Observed band size: 17 kDa

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• WB

Lab

Western blot - Anti-MUC1 antibody [EPR1023] (AB109185)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution

All lanes:

Colon cancer at 10 µg

Secondary

All lanes:

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

Predicted band size: 122 kDa

Observed band size: 24 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-MUC1 antibody [EPR1023] (AB109185)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.