Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling AKT1 + AKT2 with ab182729 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and nuclear staining on Human tonsil is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human bladder cancer tissue labeling AKT1 + AKT2 with ab182729 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and nuclear staining on Human bladder cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling AKT1 + AKT2 with ab182729 at 1/600 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling AKT1 + AKT2 with ab182729 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

AKT1/2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab182729 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab182729 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate, 10μg (Input).
Lane 2 : ab182729 IP in HeLa whole cell lysate.
Lane 3 : Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab182729 in HeLa whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>)

Predicted band size: 56 kDa

false

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling AKT1 + AKT2 with ab182729 at 1/600 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling AKT1 + AKT2 with ab182729 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and nuclear staining on Rat cerebrum is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling AKT1 + AKT2 with ab182729 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and nuclear staining on Mouse stomach is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling AKT1 + AKT2 with ab182729 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1 : 8 seconds; Lane 2 : 2 seconds.

All lanes:

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 56 kDa

Observed band size: 56 kDa

false

• WB

Supplier Data

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>) at 1/5000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 56 kDa

Observed band size: 56 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

Human AKT1 fragment recombinant protein contains aa281-480 with a His-Tag^®. Human AKT2 fragment recombinant protein contains aa282-481 with a His-Tag^®. Human AKT3 fragment recombinant protein contains aa351-479 with a His-Tag^®.

All lanes:

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>) at 1/1000 dilution

Lane 1:

Human AKT1 fragment recombinant protein at 0.02 µg

Lane 2:

Human AKT2 fragment recombinant protein at 0.02 µg

Lane 3:

Human AKT3 fragment recombinant protein at 0.02 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 56 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Western blot : Anti-AKT1 + AKT2 antibody [EPR17062] ab182729 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 56, 58 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in AKT2 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc BSA 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human AKT2 knockout MCF7 cell line (ab286253) at 20 µg

Lane 3:

A549 at 20 µg

Lane 4:

THP-1 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 56 kDa,58 kDa

Observed band size: 56 kDa,58 kDa

false

• WB

Supplier Data

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] - BSA and Azide free (AB250634)

This data was developed using ab182729, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : Lanes 1-4 : 5 seconds; Lanes 5-7 : 3 seconds.

All lanes:

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>) at 1/5000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat heart lysate at 10 µg

Lane 5:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 6:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 7:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 56 kDa

Observed band size: 56 kDa

false