Anti-Angiogenin antibody [RM2092]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Angiogenin antibody [RM2092] (AB325241)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Angiogenin with ab325241 at 1/500 (1.022 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on human liver.
The primary antibody was incubated 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 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-Angiogenin antibody [RM2092] (AB325241)

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labeling Angiogenin with ab325241 at 1/500 (1.022 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on human hepatocellular carcinoma (PMID 25567797).
The primary antibody was incubated 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 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-Angiogenin antibody [RM2092] (AB325241)

Immunohistochemical analysis of paraffin-embedded A Untreated HepG2 (human hepatocellular carcinoma epithelial cell) cell pellet B HepG2 treated with 300 ng/ml BFA for 24 hours cell pellet C Untreated Daudi (human Burkitt's lymphoma lymphoblast) cell pellet D Daudi treated with 300 ng/ml BFA for 24 hours cell pellet tissue labeling Angiogenin with ab325241 at 1/1000 (0.511 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Weak positive on image A : Untreated HepG2 cell pellet, positive staining on image B : HepG2 treated with 300 ng/ml BFA for 24 hours cell pellet, no staining on image C : Untreated Daudi cell pellet, and weak positive staining on image D : Daudi treated with 300 ng/ml BFA for 24 hours cell pellet.
The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Angiogenin antibody [RM2092] (AB325241)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) (Right magenta) HepG2 treated with brefeldin A (300ng/mL) for 24 hours (Right green) Daudi (human Burkitt's lymphoma lymphoblast) (Left magenta) Daudi treated with brefeldin A (300ng/mL) for 24 hours (Left green) cells labelling Angiogenin with ab325241 at 1/5000 dilution (0.01ug) / Green and magenta compared with a Rabbit monoclonal IgG (ab172730) (Black) and Grey isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

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

Negative control : Daudi.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Angiogenin antibody [RM2092] (AB325241)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) treated with BFA(300ng/ml) for 24hours;HepG2 cells labelling Angiogenin with ab325241 at 1/50 (9.8 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing increased cytoplasmic staining in HepG2 cells treated with BFA(300ng/ml) for 24hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/ab7291 1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325241 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• WB

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Western blot - Anti-Angiogenin antibody [RM2092] (AB325241)

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

Low expression : Daudi, Ramos

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

All lanes:

Western blot - Anti-Angiogenin antibody [RM2092] (ab325241) at 1/1000 dilution

Lane 1:

Untreated HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 treated with 300 ng/ml BFA for 24 hours whole cell lysate at 20 µg

Lane 3:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

A549 treated with 300 ng/ml BFA for 24 hours whole cell lysate at 20 µg

Lane 5:

Untreated Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 6:

Daudi treated with 300 ng/ml BFA for 24 hours whole cell lysate at 20 µg

Lane 7:

Untreated Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 8:

Ramos treated with 300 ng/ml BFA for 24 hours 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: 14 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Angiogenin antibody [RM2092] (AB325241)

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

Low expression : spleen, cerebellum

The identity of the higher MW band at approximately 75 kDa (in lane 4 ) is unknown.

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

All lanes:

Western blot - Anti-Angiogenin antibody [RM2092] (ab325241) at 1/1000 dilution

Lane 1:

Human colon tissue lysate at 40 µg

Lane 2:

human breast tissue lysate at 40 µg

Lane 3:

Human spleen tissue lysate at 40 µg

Lane 4:

Human cerebellum tissue lysate at 40 µg

Secondary

All lanes:

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

Observed band size: 14 kDa,36 kDa

false

Exposure time: 180s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Angiogenin antibody [RM2092] (AB325241)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling Angiogenin with ab325241 at 1/500 (1.022 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Low expression : weak staining on human spleen.
The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Angiogenin antibody [RM2092] (AB325241)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Daudi (human Burkitt's lymphoma lymphoblast) treated with BFA(300ng/ml) 24hours;Daudi cells labelling Angiogenin with ab325241 at 1/50 (9.8 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Low expression : Confocal image showing no staining in Daudi cells and Daudi cells treated with BFA(300ng/ml) 24hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/ab7291 1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325241 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-Angiogenin antibody [RM2092] (AB325241)

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

This antibody does not cross-react with overexpressed mouse Angiopoietin 2, Angiopoietin 3 and Angiopoietin 4 by western blot.

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

In Western blot, Anti-6X His tag® antibody [AD1.1.10] (ab15149) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Angiogenin antibody [RM2092] (ab325241) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human Angiogenin expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse Angiopoietin 2 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a mouse Angiopoietin 3 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a mouse Angiopoietin 4 expression vector containing a myc-His-tag®, 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: 16 kDa,36 kDa

false

Exposure time: 15s