重组Anti-RAB27B抗体[EPR28647-45] (ab317264)

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

Negative control: A-673

Low expression: Huh7(PMID: 22553406)

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

Exposure time: Lane1-2: 15 seconds, lane3-4: 180 seconds

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

Low expression liver(PMID: 14625374)A20, F9

Lane 3-4 were developed using a higher sensitivity ECL substrate.

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

Exposure time: Lane1-2: 15 seconds, lane3-4: 180 seconds

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

Low expression: liver(PMID: 14625374)

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

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human stomach (PMID: 22811270). The section was incubated with ab317264 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 stomach tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse stomach. The section was incubated with ab317264 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 RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on megakaryocytes and platelets in mouse spleen. The section was incubated with ab317264 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 Rat stomach tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat stomach. The section was incubated with ab317264 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 Rat spleen tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on megakaryocytes and platelets in rat spleen. The section was incubated with ab317264 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 liver tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression: positive staining on platelets of human liver. The section was incubated with ab317264 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 liver tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression: positive staining on platelets of mouse liver. The section was incubated with ab317264 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 Rat liver tissue labeling RAB27B with ab317264 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression: positive staining on platelets of rat liver. The section was incubated with ab317264 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 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse stomach (fresh frozen) tissue labeling RAB27B with ab317264 at 1/500 (1.018 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining on epithelium of mouse stomach. The nuclear counterstain was DAPI (Blue). The section was incubated with ab317264 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh frozen) tissue labeling RAB27B with ab317264 at 1/500 (1.018 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Low expression: confocal image showing no staining on mouse liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab317264 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell) cells labelling RAB27B with ab317264 at 1/50 (10.18 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 U-87 MG cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control: A-673.
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 U-87 MG (human glioblastoma-astrocytoma epithelial cell) (Right) / A-673 (human muscle Ewings sarcoma cell line) (Left) cells labelling RAB27B with ab317264 at 1/5000 dilution (0.01ug) (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.

Negative control: A-673

RAB27B was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate with ab317264 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317264 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 2: ab317264 IP in U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317264 in U-87 MG whole cell lysate

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

Exposure time: 6 seconds.

Dot blot analysis of RAB27B using ab317264 at 1:1000 (0.509 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: His-tagged Human RAB27B protein
Lane 2: Human RAB27A protein
Lane 3: His-tagged Human RAB3A protein

Exposure time: 180 seconds.

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

This antibody doesn't cross-react with Human RAB27A and RAB3A.