Anti-Rab3A 抗体 [EPR26022-9] (ab302518)

Anti-RAB3A antibody [EPR26022-9] (ab302518) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab302518 was shown to bind specifically to RAB3A. A band was observed at 27 kDa in wild-type SKNFI cell lysates with no signal observed at this size in RAB3A knockout cell line. To generate this image, wild-type and RAB3A knockout SKNFI cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Loading control: Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1:200000) (36KDa)

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

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Rab3A-KO homozygous mice (Strain ID: T016905).

Blocking and dilution buffer: 5% NFDM/TBST. 

Negative controls: liver, skeletal muscle (PMID: 11865063).

Bands above 250 kDa may be Rab3A aggregates. 

Immunohistochemical analysis of paraffin-embedded (A) Cerebellum tissue from wild-type C57BL/6JGpt mice (B) Cerebellum tissue from Rab3A knockout mice labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Sections were fixed and
counterstained with Hematoxylin. Antigen retrieval was Heat mediated with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

Positive staining on (A) Cerebellum tissue from wild-type C57BL/6JGpt mice and no staining on (B) Cerebellum tissue from Rab3A knockout mice.
The section was incubated with ab302518 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Rab3A-KO homozygous mice (Strain ID: T016905).

Immunohistochemical analysis of paraffin-embedded (A) Brain cortex tissue from wild-type C57BL/6JGpt mice (B) Brain cortex tissue from Rab3A knockout mice labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Sections were fixed and counterstained with Hematoxylin. Antigen retrieval was Heat mediated with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

Positive staining on (A) Brain cortex tissue from wild-type C57BL/6JGpt mice and no staining on (B) Brain cortex tissue from Rab3A knockout mice.
The section was incubated with ab302518 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Rab3A-KO homozygous mice (Strain ID: T016905).

Blocking and dilution buffer: 5% NFDM/TBST. 

Bands above 150 kDa may be Rab3A aggregates.

Blocking and dilution buffer: 5% NFDM/TBST. 

Bands above 150 kDa may be Rab3A aggregates.

Exposure time: 5.5 seconds.

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Cytoplasmic staining on human cerebellum. The section was incubated with ab302518 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: A ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Cytoplasmic staining on human cerebrum is observed (PMID: 26076492). The section was incubated with ab302518 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: A ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Cytoplasmic staining on mouse cerebellum (PMID: 10407024). The section was incubated with ab302518 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: A ready to use LeicaDS9800 (BOND^® Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Cytoplasmic staining on rat cerebellum is observed (PMID: 12937130). The section was incubated with ab302518 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: A ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling Rab3A with ab302518 at 1/5000 dilution (0.11 µg/ml) followed by a ready to use LeicaDS9800 (BOND^® Polymer Refine Detection). The section was incubated with ab302518 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Negative control: No staining on human skeletal muscle is observed.

Secondary antibody only control: A ready to use LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebellum (fresh) tissue labeling Rab3A with ab302518 at 1/500 (1.1 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml) (Green). Positive staining on mouse cerebellum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/ml). 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebellum (fresh) tissue labeling Rab3A with ab302518 at 1/500 dilution (1.1 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml) (Green). Positive staining on rat cerebellum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labeling Rab3A with ab302518 at 1/50 dilution (11.0 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing cytoplasmic staining in SH-SY5Y cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5µg/ml) (Red). 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 dilution (2 µg/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labeling Rab3A with ab302518 at 1/50 dilution (11.0 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at dilution 1/1000 (2µg/ml) (Green). Confocal image showing cytoplasmic staining in Neuro-2a cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5µg/ml) (Red). 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 dilution (2µg/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma small irregularly shaped cells) cells labeling Rab3A with ab302518 at 1/50 dilution (11.0 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing cytoplasmic staining in PC-12 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml) (Red). 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 dilution (2 µg/ml).

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labeling Rab3A with ab302518 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labeling Rab3A with ab302518 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized PC-12 (rat adrenal gland pheochromocytoma) cells labeling Rab3A with ab302518 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Rab3A was immunoprecipitated from 0.35 mg human cerebellum tissue lysate with ab302518 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302518 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: human cerebellum tissue lysate 10 µg (Input)

Lane 2: ab302518 IP in human cerebellum tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302518 in human cerebellum tissue lysate

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 seconds.

Observed MW (kDa): 25, 45.

45 kDa band may represent Rab3A dimer (PMID: 9252412).

Rab3A was immunoprecipitated from 0.35 mg mouse cerebellum tissue lysate with ab302518 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302518 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: mouse cerebellum tissue lysate 10 µg (Input)

Lane 2: ab302518 IP in mouse cerebellum tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302518 in mouse cerebellum tissue lysate

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 seconds.

Observed MW (kDa): 25, 45.

45 kDa band may represent Rab3A dimer (PMID: 9252412).

Dot blot analysis of Rab3A using ab302518 at 1/1000 dilution (0.55 μg/ml), followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Lane 1: HEK-293 transfected with human Rab3A (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 2: HEK-293 transfected with human Rab3B (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3: HEK-293 transfected with human Rab3C (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 4: HEK-293 transfected with human Rab3D (WT) expression vector containing a myc-His-tag®, whole cell lysate

Exposure time: 3 minutes.

Blocking and diluting buffer: 5% NFDM/TBST.