Anti-CLUH antibody [EPR28732-85]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded A Wild-type HEK-293 (human embryonic kidney epithelial cell) cell pellet B CLUH knockout HEK-293 whole cell pellet tissue labeling CLUH with ab317693 at 1/100 (5.15 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Positive staining on (A) Wild-type HEK-293 cell pellet, no staining on (B) CLUH knockout HEK-293 whole cell pellet. The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling CLUH with ab317693 at 1/100 (5.15 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Low expression : almost no staining on human lung. The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling CLUH with ab317693 at 1/100 (5.15 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Positive staining on human kidney. The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

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-CLUH antibody [EPR28732-85] (AB317693)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK293T (human embryonic kidney epithelial cell) cells labelling CLUH with ab317693 at 1/50 (10.3 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 cytoplasmic staining in HEK293T cell line (shown in green) and no staining in CLUH KO HEK293T cell line. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). 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 (Magenta). 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-CLUH antibody [EPR28732-85] (AB317693)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized CLUH KO HEK293T (human embryonic kidney epithelial cell, Left) / Parental HEK293T (Right) cells labelling CLUH with ab317693 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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling CLUH with ab317693 at 1/50 (10.3 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 cytoplasmic staining in NIH/3T3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). 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 (Magenta). 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-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling CLUH with ab317693 at 1/1000 (0.515 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Positive staining on rat liver. The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling CLUH with ab317693 at 1/1000 (0.515 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Low expression : no staining on mouse lung (PMID : 28188211). The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling CLUH with ab317693 at 1/1000 (0.515 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Low expression : no staining on rat lung. The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling CLUH with ab317693 at 1/50 dilution (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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLUH antibody [EPR28732-85] (AB317693)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling CLUH with ab317693 at 1/1000 (0.515 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Positive staining on mouse liver (PMID : 32149416). The section was incubated with ab317693 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

Supplier Data

Western blot - Anti-CLUH antibody [EPR28732-85] (AB317693)

In Western blot, ab317693 was shown to bind specifically to CLUH. Target of interest was observed at 160 kDa in wild-type HEK-293 cell lysates (lane 1) with no signal observed at this size in CLUH knockout cell line (lane 2) (knockout cell line ab266407 / knockout cell lysate ab258367).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CLUH antibody [EPR28732-85] (ab317693) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

CLUH knockout HEK-293 whole cell lysate at 20 µg

Lane 3:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma cell) 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 1/20000 dilution

Observed band size: 160 kDa,36 kDa

false

Exposure time: 103s

• WB

Supplier Data

Western blot - Anti-CLUH antibody [EPR28732-85] (AB317693)

Low expression : lung (PMID : 28188211).

The identity of the lower MW band at approximately 37 kDa (in lane 1) is unknown.

Lanes 1-4 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and lane 5 is applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-4 : 103 seconds; Lane 5 : 158 seconds.

All lanes:

Western blot - Anti-CLUH antibody [EPR28732-85] (ab317693) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Rat liver tissue lysate at 20 µg

Lane 4:

Rat lung tissue lysate at 20 µg

Lane 5:

Human kidney tissue lysate at 20 µg

Secondary

Lanes 1 - 4:

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

Lane 5:

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

Observed band size: 160 kDa,36 kDa

false