Anti-MUPP1 antibody [EPR26317-59]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A549 (human lung carcinoma epithelial cell) cells labelling MUPP1 with ab302621 at 1/500 dilution (0.1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscl tissue labeling MUPP1 with ab302621 at 1/800 (0.683 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Negative control : no staining in mouse skeletal muscle (PMID : 12706259). The section was incubated with ab302621 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval 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-MUPP1 antibody [EPR26317-59] (AB302621)

Immunohistochemical analysis of paraffin-embedded Rat choroid plexus tissue labeling MUPP1 with ab302621 at 1/800 (0.683 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in rat choroid plexus (PMID : 30518636, PMID : 12706259 ). The section was incubated with ab302621 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval 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-MUPP1 antibody [EPR26317-59] (AB302621)

Immunohistochemical analysis of paraffin-embedded Mouse choroid plexus tissue labeling MUPP1 with ab302621 at 1/800 (0.683 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in mouse choroid plexus (PMID : 30518636, PMID : 12706259). The section was incubated with ab302621 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat choroid plexus tissue labeling MUPP1 with ab302621 at 1/50 (10.92 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Positive staining on rat choroid plexus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mIMCD3 (mouse inner medullary collecting duct epithelial cell) cells labelling MUPP1 with ab302621 at 1/50 (10.92 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing membranous and cytoplasmic staining in mIMCD3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse choroid plexus tissue labeling MUPP1 with ab302621 at 1/50 (10.92 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Positive staining on mouse choroid plexus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling MUPP1 with ab302621 at 1/800 (0.683 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Negative control : no staining in rat skeletal muscle (PMID : 12706259). The section was incubated with ab302621 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized mIMCD3 (mouse inner medullary collecting duct epithelial cell) cells labelling MUPP1 with ab302621 at 1/500 dilution (0.1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

MUPP1 was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell), whole cell lysate 10 µg with ab302621 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302621 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate 10 µg

Lane 2 : ab302621 IP in C6 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab302621 in C6 whole cell lysate

Blocking and dilution 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.

The bands beneath the target band are likely to be degraded target fragments.

All lanes:

Immunoprecipitation - Anti-MUPP1 antibody [EPR26317-59] (ab302621) at 1/30 dilution

All lanes:

C6 (rat glial tumor glial cell), whole cell lysate

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

Predicted band size: 219 kDa

true

Exposure time: 58s

• WB

Lab

Western blot - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Western blot : Anti-MPDZ antibody [EPR26317-59] (ab302621) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab302621 was shown to bind specifically to MPDZ. A band was observed at 268 kDa in wild-type A549 cell lysates with no signal observed at this size in MPDZ knockout cell line. To generate this image, wild-type and MPDZ knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-MUPP1 antibody [EPR26317-59] (ab302621) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MPDZ knockout A549 cell lysate at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Lane 4:

SW480 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 268 kDa

false

• WB

Supplier Data

Western blot - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 12403818). Lysates in lane1-4 were freshly made and used for Western blotting immediately to minimize protein degradation. The bands beneath the target band are likely to be degraded target fragments. Samples are non-boiled as boiling may cause protein aggregates. Exposure time : Lane 1-7 : 3 minutes Lane 8 : 26 seconds

All lanes:

Western blot - Anti-MUPP1 antibody [EPR26317-59] (ab302621) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell), whole cell fresh lysate 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell fresh lysate 20 µg

Lane 3:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell fresh lysate 20 µg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell), whole cell fresh lysate 20 µg

Lane 5:

C6 (rat glial tumor glial cell), whole cell lysate 40 µg

Lane 6:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate 40 µg

Lane 7:

human cerebellum tissue lysate 40 µg

Lane 8:

mIMCD3 (mouse inner medlary collecting duct epithelial cell), whole cell lysate 40 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

Observed band size: 280 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-MUPP1 antibody [EPR26317-59] (AB302621)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 12403818). Low expression : skeletal muscle (PMID : 12706259) Lysate in lane 5 was freshly made and used for Western blotting immediately to minimize protein degradation. The bands beneath the target band are likely to be degraded target fragments. Samples are non-boiled as boiling may cause protein aggregates. Exposure time : Lane 1-4 : 26 seconds Lane 5 : 3 minutes

All lanes:

Western blot - Anti-MUPP1 antibody [EPR26317-59] (ab302621) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate 40 µg

Lane 2:

Mouse skeletal muscle tissue lysate 40 µg

Lane 3:

Rat brain tissue lysate 40 µg

Lane 4:

Rat skeletal muscle tissue lysate 40 µg

Lane 5:

Mouse testis tissue lysate 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

Observed band size: 280 kDa,278 kDa

false

Exposure time: 26s