重组Anti-CLUH抗体[EPR28732-85] (ab317693)

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

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.