Recombinant Anti-AKT3 + AKT2 + AKT1 antibody [RM1043] (ab300473)

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

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

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/2000, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human cerebrum is observed. The section was incubated with ab300473 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection). 

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

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/2000, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast carcinoma is observed. The section was incubated with ab300473 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection). 

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

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/2000, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse cerebrum is observed. The section was incubated with ab300473 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection). 

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

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/2000, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat cerebrum is observed. The section was incubated with ab300473 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection). 

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

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/2000, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Low expression on human skeletal muscle is observed. The section was incubated with ab300473 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. 

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection). 

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

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebral cortex (fresh) tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/100 dilution  (4.4 μg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preabsorbed at 1/1000 dilution (2 µg/mL)  (Green). Positive staining on mouse cerebral cortex is observed. The nuclear counterstain was DAPI (Blue).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebral cortex (fresh) tissue labeling AKT1 + AKT2 + AKT3  with ab300473 at 1/100 dilution  (4.4 μg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preabsorbed at 1/1000 dilution (2 µg/mL)  (Green). Positive staining on rat cerebral cortex is observed. The nuclear counterstain was DAPI (Blue).

Immunofluorescent analysis of 4% paraformaldehyde fixed and 0.1% TritonX-100 permeabilized MCF7 (human breast adenocarcinoma epithelial cell）labeling AKT1 + AKT2 + AKT3 with ab300743 at 1/50 dilution (8.8 μg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Green) preadsorbed at 1:1000 (2 μg/mL). Confocal image showing nuclear and cytoplasmic staining in MCF7 cell line. 

Ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (Red) was used as a counterstain at 1:200 dilution (2.5 μg/ml). Nuclear counter satin is DAPI. 

Immunofluorescent analysis of 4% paraformaldehyde fixed and 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) labeling AKT1 + AKT2 + AKT3 with ab300743 at 1/50 dilution (8.8 μg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Green) preadsorbed at 1:1000 (2 μg/mL). Confocal image showing nuclear and cytoplasmic staining in NIH/3T3 cell line. 

Ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (Red) was used as a counterstain at 1:200 dilution (2.5 μg/ml). Nuclear counter satin is DAPI. 

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methanol permeabilized MCF7 (human breast adenocarcinoma epithelial cell) cells labelling AKT1 + AKT2 + AKT3 with ab300473 at 1/500 dilution (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.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling AKT1 + AKT2 + AKT3 with ab300473 at 1/500 dilution (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.

AKT1 + AKT2 + AKT3 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg with ab300473 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300473 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg (Inset)

Lane 2: ab300114 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300473 in NIH/3T3 whole cell lysate

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

Exposure time: 6 seconds. 

AKT1 + AKT2 + AKT3 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab300473 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300473 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg (Inset)

Lane 2: ab300114 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300473 in HeLa whole cell lysate

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

Exposure time: 6 seconds.