Anti-Granzyme A antibody [RM2061]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme A antibody [RM2061] (AB322896)

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling Granzyme A with ab322896 at 1/1000 (0.493 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in immune cells of human endometrium.
The section was incubated with ab322896 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).

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-Granzyme A antibody [RM2061] (AB322896)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Upper Left) / 293T (human embryonic kidney epithelial cell) transfected with an Granzyme A expression vector containing a myc-His-tag® (Upper Middle) / 293T cells transfected with an empty expression vector containing a myc-His-tag® (Upper Right) / 293T cells transfected with homologous human Granzyme expression vector (GrzmB, GrzmH, GrzmK, GrzmM) containing a myc-His-tag® (Lower). cells labelling Granzyme A with ab322896 at 1/50 dilution (1ug) compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

After fixation and permeability, cells were stained with anti-myc tag conjugated to Alexa Fluor® 647.
Crossreactivity with protein Granzyme B, Granzyme H, Granzyme K, Granzyme M were fully tested.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme A antibody [RM2061] (AB322896)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling Granzyme A with ab322896 at 1/1000 (0.493 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in immune cells of human ovarian cancer.
The section was incubated with ab322896 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).

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-Granzyme A antibody [RM2061] (AB322896)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC (Human primary peripheral blood mononuclear cell) cells labelling Granzyme A with ab322896 at 1/4000 (0.123 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in subsets of human PBMCs (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).

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 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Granzyme A antibody [RM2061] (AB322896)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human PBMC(human peripheral blood mononuclear cell) cells labelling Granzyme A with ab322896 at 1/5000 dilution (0.01 ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Cells were surface stained with anti-CD14 conjugated to Alexa Fluor®647. Then fixed with 2% PFA for 10min followed by intracellularly stained with isotype control (ab172730) or the antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Granzyme A antibody [RM2061] (AB322896)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human PBMC(human peripheral blood mononuclear cell) cells labelling Granzyme A with ab322896 at 1/5000 dilution (0.01 ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Cells were surface stained with anti-CD8 conjugated to Pacific Blue. Then fixed with 2% PFA for 10min followed by intracellularly stained with isotype control (ab172730) or the antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme A antibody [RM2061] (AB322896)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling Granzyme A with ab322896 at 1/1000 (0.493 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human spleen.
The section was incubated with ab322896 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).

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-Granzyme A antibody [RM2061] (AB322896)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) cells labelling Granzyme A with ab322896 at 1/4000 (0.123 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in No-GFP-CD16.NK-92 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : 293T.
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 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Granzyme A antibody [RM2061] (AB322896)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human PBMC(human peripheral blood mononuclear cell) cells labelling Granzyme A with ab322896 at 1/5000 dilution (0.01 ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Cells were surface stained with anti-CD56 conjugated to PE. Then fixed with 2% PFA for 10min followed by intracellularly stained with isotype control (ab172730) or the antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Granzyme A antibody [RM2061] (AB322896)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NK-92(human malignant non-Hodgkin's lymphoma natural killer cell, Right) / 293T(human embryonic kidney epithelial cell, Left) cells labelling Granzyme A with ab322896 at 1/5000 dilution (0.01 ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left 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 : 293T.

• IP

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Immunoprecipitation - Anti-Granzyme A antibody [RM2061] (AB322896)

Granzyme A was immunoprecipitated from 0.35 mg No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate with ab322896 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322896 at 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate
Lane 2 : ab322896 IP in No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322896 in No-GFP-CD16.NK-92 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-Granzyme A antibody [RM2061] (ab322896) at 1/30 dilution

All lanes:

No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) 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

false

Exposure time: 3s

• WB

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Western blot - Anti-Granzyme A antibody [RM2061] (AB322896)

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

Negative control : 293T, HeLa.

Negative control : cerebellum.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 10411926).

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

Exposure time : Lanes 1-3 : 1 second, lanes 4-5 : 180 seconds

All lanes:

Western blot - Anti-Granzyme A antibody [RM2061] (ab322896) at 1/1000 dilution

Lane 1:

No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

Human spleen tissue lysate at 20 µg

Lane 5:

Human cerebellum tissue 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 1/100000 dilution

Observed band size: 24 kDa,26 kDa,36 kDa

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme A antibody [RM2061] (AB322896)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Granzyme A with ab322896 at 1/1000 (0.493 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining in human cerebrum.
The section was incubated with ab322896 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).

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