Anti-EZHIP antibody [EPR28396-50]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EZHIP antibody [EPR28396-50] (AB313392)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscl tissue labeling CXorf67 with ab313392 at 1/200 (2.555 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control : No staining on human skeletal muscle.The section was incubated with ab313392 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). 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-EZHIP antibody [EPR28396-50] (AB313392)

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling CXorf67 with ab313392 at 1/200 (2.555 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control : No staining on human cardiac muscle.The section was incubated with ab313392 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). 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-EZHIP antibody [EPR28396-50] (AB313392)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling CXorf67 with ab313392 at 1/200 (2.555 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human testis (PMID : 31451685).The section was incubated with ab313392 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). 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-EZHIP antibody [EPR28396-50] (AB313392)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling CXorf67 with ab313392 at 1/200 (2.555 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human placenta.The section was incubated with ab313392 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EZHIP antibody [EPR28396-50] (AB313392)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-2 OS (human bone osteosarcoma epithelial cell) cells labelling CXorf67 with ab313392 at 1/500 (1.022 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 nuclear staining in U-2 OS cell line.Low expression : 293T (PMID : 34880314).Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-EZHIP antibody [EPR28396-50] (AB313392)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (human embryonic kidney epithelial cell, Left) / U-2 OS (human bone osteosarcoma epithelial cell, Right) cells labelling EZHIP with ab313392 at 1/500 dilution (0.1 ug)/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 1/5000 dilution was used as the secondary antibody. Low expression : 293T (PMID : 34880314).

• WB

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Western blot - Anti-EZHIP antibody [EPR28396-50] (AB313392)

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

Low expression : 293T (PMID : 34880314).

The molecular weight observed is consistent with what has been described in the literature (PMID : 34880314).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

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

All lanes:

Western blot - Anti-EZHIP antibody [EPR28396-50] (ab313392) at 1/1000 dilution

Lane 1:

U-2OS (human bone osteosarcoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 50 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

Observed band size: 50 kDa,75 kDa

false

Exposure time: 92s

• WB

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Western blot - Anti-EZHIP antibody [EPR28396-50] (AB313392)

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- H3 antibody (ab176842) loading control staining at 1/100000 dilution.

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

All lanes:

Western blot - Anti-EZHIP antibody [EPR28396-50] (ab313392) at 1/1000 dilution

Lane 1:

Mouse testis cytoplasmic fraction at 50 µg

Lane 2:

Mouse testis nuclear fraction at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 75 kDa

true

Exposure time: 180s