Anti-AKT2 antibody [4H7]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT2 antibody [4H7] (AB175354)

Immunohistochemical analysis of deparaffinized Human Esophageal cancer tissue labeling AKT2 with ab175354 at 1/200 dilution. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-AKT2 antibody [4H7] (AB175354)

Immunofluorescent analysis of AKT2 (green) showing staining in the cytoplasm and nucleus of MCF-7 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an AKT2 monoclonal antibody (ab175354) in 3% BSA-PBS at a dilution of 1 : 20 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT2 antibody [4H7] (AB175354)

Immunohistochemical analysis of deparaffinized normal Human Medulla Oblongata tissue labeling AKT2 with ab175354 at 1/200 dilution. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-AKT2 antibody [4H7] (AB175354)

Immunofluorescent analysis of AKT2 (green) showing staining in the cytoplasm and nucleus of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an AKT2 monoclonal antibody (ab175354) in 3% BSA-PBS at a dilution of 1 : 20 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

• ChIP

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ChIP - Anti-AKT2 antibody [4H7] (AB175354)

Chromatin immunoprecipitation analysis of Akt1 and Akt2 was performed using cross-linked chromatin from 1 x 10⁶ HCT116 colon carcinoma cells treated with serum for 0, 15, 30, and 60 minutes. Immunoprecipitation was performed with 1.0ul/100ul well volume of an Atk1 monoclonal antibody and an Akt2 monoclonal antibody (ab175354). Chromatin aliquots from ~1 x 10⁵ cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1ul of eluted DNA in 2ul SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions); the zigzag line represents an intron; and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-AKT2 antibody [4H7] (AB175354)

Immunofluorescent analysis of AKT2 (green) showing staining in the cytoplasm and nucleus of C2C12 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an AKT2 monoclonal antibody (ab175354) in 3% BSA-PBS at a dilution of 1 : 20 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

• IP

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Immunoprecipitation - Anti-AKT2 antibody [4H7] (AB175354)

Immunoprecipitation of AKT2 was performed on HeLa cells. The antigen : antibody complex was formed by incubating 750 μg whole cell lysate with 2 μg of ab175354. WB detection used ab175354 at 1/1000 dilution.

All lanes:

Immunoprecipitation - Anti-AKT2 antibody [4H7] (ab175354)

Predicted band size: 55 kDa

true

• WB

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Western blot - Anti-AKT2 antibody [4H7] (AB175354)

All lanes:

Western blot - Anti-AKT2 antibody [4H7] (ab175354) at 1/1000 dilution

Lane 1:

Non-transfected U2OS cells

Lane 2:

U2OS cells transfected with AKT2 siRNA

Secondary

All lanes:

goat anti-mouse-HRP at 1/20000 dilution

Predicted band size: 55 kDa

false

• WB

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Western blot - Anti-AKT2 antibody [4H7] (AB175354)

All lanes:

Western blot - Anti-AKT2 antibody [4H7] (ab175354) at 1/1000 dilution

Lane 1:

MCF7 whole cell lysate at 25 µg

Lane 2:

HeLa whole cell lysate at 25 µg

Lane 3:

HepG2 whole cell lysate at 25 µg

Lane 4:

A549 whole cell lysate at 25 µg

Lane 5:

293T whole cell lysate at 25 µg

Lane 6:

Jurkat whole cell lysate at 25 µg

Lane 7:

A431 whole cell lysate at 25 µg

Lane 8:

U2OS whole cell lysate at 25 µg

Lane 9:

COS7 whole cell lysate at 25 µg

Lane 10:

3T3 L1 whole cell lysate at 25 µg

Lane 11:

NRK whole cell lysate at 25 µg

Secondary

All lanes:

goat anti-mouse-HRP at 1/20000 dilution

Predicted band size: 55 kDa

true