重组Anti-Granzyme B抗体[RM1165] (ab317458)

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

Negative control: Jurkat (PMID:18437383).

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade  (ab176842) staining at 1/100000 dilution.

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

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).

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

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

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

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

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

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 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).

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).

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.

Exposure time: 180 seconds.

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.

Exposure time: 41 seconds.