Anti-MAdCAM1 antibody [EPR27223-77]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MAdCAM1 antibody [EPR27223-77] (AB307827)

Immunohistochemical analysis of paraffin-embedded mouse jejunum tissue labeling MAdCAM1 with ab307827 at 1/500 dilution (1.084 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on the high endothelial venules of peripheral lymph nodes in mouse jejunum. The section was incubated with ab307827 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MAdCAM1 antibody [EPR27223-77] (AB307827)

Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labeling MAdCAM1 with ab307827 at 1/500 dilution (1.084 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Negative control : No staining on mouse cardiac muscle. The section was incubated with ab307827 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-MAdCAM1 antibody [EPR27223-77] (AB307827)

MAdCAM1 was immunoprecipitated from 0.35 mg mouse colon tissue lysate 10 ug with ab307827 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307827 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : mouse colon tissue lysate 10 ug Lane 2 : ab307827 IP in mouse colon tissue lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307827 in mouse colon tissue lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-MAdCAM1 antibody [EPR27223-77] (ab307827) at 1/30 dilution

All lanes:

ab307827 IP in mouse colon tissue lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

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Western blot - Anti-MAdCAM1 antibody [EPR27223-77] (AB307827)

This antibody does not cross-react with human MAdCAM1. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution, anti-GAPDH antibody (ab181602) loading control staining at 1/200, 000 dilution. Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MAdCAM1 antibody [EPR27223-77] (ab307827) at 1/1000 dilution

Lane 1:

HEK-293 transfected with an empty vector (vector control), containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

HEK-293 transfected with a human MAdCAM1 (WT) expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

HEK-293 transfected with a mouse MAdCAM1 (WT) expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Predicted band size: 40 kDa

Observed band size: 60 kDa

false

• WB

Supplier Data

Western blot - Anti-MAdCAM1 antibody [EPR27223-77] (AB307827)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-MAdCAM1 antibody [EPR27223-77] (ab307827) at 1/1000 dilution

Lane 1:

Untreated bEnd.3 whole cell lysate at 20 µg

Lane 2:

TNF alpha treated bEnd.3 whole cell 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 1/100000 dilution

Predicted band size: 40 kDa

Observed band size: 60 kDa

true