Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling Rab 6A with ab271094 at 1/2000 (0.296 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Granular cytoplasmic staining on mouse testis. The section was incubated with ab271094 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized Neuro-2a cells labelling Rab 6A with ab271094 at 1/50 (11.84 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing golgi staining in Neuro-2a cell line. ab169276 Anti-GM130 mouse polyclonal antibody - cis-Golgi Marker was used to counterstain GM130 at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

All lanes : Anti-Rab 6A antibody [EPR24472-24] (ab271094) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 4 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 5 : A549 (human lu carcinoma epithelial cell) whole cell lysate
Lane 6 : Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate
Lane 7 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 8 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 9 : C6 (rat glial tumor glial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 24 kDa
Observed band size: 24 kDa

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

Exposure time: 26 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (Human lung carcinoma epithelial cell) cells labelling Rab 6A with ab271094 at 1/50 dilution (1ug) (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). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Rab 6A was immunoprecipitated from 0.35 mg Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate with ab271094 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271094 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate 10 ug

Lane 2: ab271094 IP in Neuro-2a whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271094 in Neuro-2a whole cell lysate

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

Exposure time: 10 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling Rab 6A with ab271094 at 1/2000 (0.296 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Granular cytoplasmic staining on human spleen. The section was incubated with ab271094 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized A549 cells labelling Rab 6A with ab271094 at 1/50 (11.84 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing golgi staining in A549 cell line. ab169276 Anti-GM130 mouse polyclonal antibody - cis-Golgi Marker was used to counterstain the GM-130 at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

All lanes : Anti-Rab 6A antibody [EPR24472-24] (ab271094) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Human heart tissue lysate
Lane 3 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 24 kDa
Observed band size: 24 kDa

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

Exposure time: Lane 1-2: 15 secondsLane 3: 48 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling Rab 6A with ab271094 at 1/50 dilution (1ug) (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). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-Rab 6A antibody [EPR24472-24] (ab271094) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 24 kDa
Observed band size: 24 kDa

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

Exposure time: 48 seconds.

 

All lanes : Anti-Rab 6A antibody [EPR24472-24] (ab271094) at 1/1000 dilution

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat heart tissue lysate
Lane 3 : Rat kidney tissue lysate
Lane 4 : Rat spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 24 kDa
Observed band size: 24 kDa

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

Exposure time: Lane 1, 3, 4: 48 secondsLane 2: 3 minutes.

 

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labelling Rab 6A with ab271094 at 1/2000 (0.296 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Granular cytoplasmic staining on rat spleen. The section was incubated with ab271094 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Rab 6A was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate with ab271094 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271094 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A549 (human lung carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab271094 IP in A549 whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab271094 in A549 whole cell lysate

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

Exposure time: 10 seconds.