Anti-Granzyme B antibody [RM1165] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Granzyme B with ab317458 at 1/1000 (0.51 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab317458 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized KARPAS-299 (human T cell lymphoma cell) cells labelling Granzyme B with ab317458 at 1/50 (10.2 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 KARPAS-299 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : Jurkat (PMID : 18437383).
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 2.5 ug/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)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human PBMC(human peripheral blood mononuclear cell) treated with 10ug/ml CD3 for 72hours and 10ug/ml CD28 for 72 hours(Bottom left and Bottom right) / Untreated Human PBMC(Top left and Top right) cells labelling Granzyme B with ab317458 at 1/50 dilution (1 ug)/Top right and Bottom right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

human PBMC are co-stained with CD8a conjugated Pacific Blue (440/50BP).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Granzyme B with ab317458 at 1/1000 (0.51 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of human colon. The section was incubated with ab317458 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

• IP

Supplier Data

Immunoprecipitation - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Granzyme B was immunoprecipitated from 0.35 mg KARPAS-299 (human T cell lymphoma cell) whole cell lysate with ab317458 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317458 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : KARPAS-299 (human T cell lymphoma cell) whole cell lysate
Lane 2 : ab317458 IP in KARPAS-299 (human T cell lymphoma cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317458 in KARPAS-299 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-Granzyme B antibody [RM1165] (<a href='/en-us/products/primary-antibodies/granzyme-b-antibody-rm1165-ab317458'>ab317458</a>) at 1/30 dilution

Lane 1:

KARPAS-299 (human T cell lymphoma cell) whole cell lysate

Lane 2:

<a href='/en-us/products/primary-antibodies/granzyme-b-antibody-rm1165-ab317458'>ab317458</a> IP in KARPAS-299 (human T cell lymphoma cell) 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: 180s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Mouse PBMC(mouse peripheral blood mononuclear cell) treated with 2.5ug/ml Concanavalin A for 72h (Bottom left and Bottom right) / Untreated mouse PBMC(Top left and Top right) cells labelling Granzyme B with ab317458 at 1/50 dilution (1 ug)/Top right and Bottom right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

mouse PBMC are co-stained with CD8a conjugated Pacific Blue (440/50BP).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Granzyme B with ab317458 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen. The section was incubated with ab317458 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Granzyme B with ab317458 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of mouse lung. The section was incubated with ab317458 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

• IP

Supplier Data

Immunoprecipitation - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Granzyme B was immunoprecipitated from 0.35 mg Mouse spleen cell treated with 2.5 ug/ml Concanavalin A for 72h whole cell lysate with ab317458 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317458 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse spleen cell treated with 2.5 ug/ml Concanavalin A for 72h whole cell lysate
Lane 2 : ab317458 IP in Mouse spleen cell treated with 2.5 ug/ml Concanavalin A for 72h whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317458 in mouse spleen cell treated with 2.5 ug/ml Concanavalin A for 72h whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-Granzyme B antibody [RM1165] (<a href='/en-us/products/primary-antibodies/granzyme-b-antibody-rm1165-ab317458'>ab317458</a>) at 1/30 dilution

Lane 1:

Mouse spleen cell treated with 2.5 ug/ml Concanavalin A for 72h whole cell lysate

Lane 2:

<a href='/en-us/products/primary-antibodies/granzyme-b-antibody-rm1165-ab317458'>ab317458</a> IP in Mouse spleen cell treated with 2.5 ug/ml Concanavalin A for 72h 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: 41s

• WB

Supplier Data

Western blot - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

Negative control : Jurkat (PMID : 18437383).

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

All lanes:

Western blot - Anti-Granzyme B antibody [RM1165] (<a href='/en-us/products/primary-antibodies/granzyme-b-antibody-rm1165-ab317458'>ab317458</a>) at 1/1000 dilution

Lane 1:

KARPAS-299 (human T cell lymphoma cell) whole cell lysate at 20 µg

Lane 2:

SR (human pleural effusion lymphoblast) whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) 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: 28 kDa,15 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Granzyme B antibody [RM1165] - BSA and Azide free (AB317459)

This data was developed using ab317458, the same antibody clone in a different buffer formulation.

The molecular weight observed is consistent with what has been described in the literature (PMID : 8663264 and 17116752).

Granzyme B forms complex with its protease inhibitor (PMID : 9774654).

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

All lanes:

Western blot - Anti-Granzyme B antibody [RM1165] (<a href='/en-us/products/primary-antibodies/granzyme-b-antibody-rm1165-ab317458'>ab317458</a>) at 1/1000 dilution

Lane 1:

Untreated mouse splenocyte lysate at 20 µg

Lane 2:

Mouse splenocytes treated with 2.5µg/ml concanavalin for 72 hours 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: 28 kDa,36 kDa

false

Exposure time: 15s