AKT Activation Antibody Sampler Kit
cat.: HAK21112
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: O15530 Human | Q9Z2A0 Mouse | O55173 Rat | P60484 Human | O08586 Mouse | O54857 Rat | P31749 Human | P31749 Human | P31751 Human | Q9Y243 Human | P31750 Mouse | Q60823 Mouse | Q9WUA6 Mouse | P47196 Rat | P47197 Rat | Q63484 Rat | P31749 Human | P31751 Human | Q9Y243 Human | P31750 Mouse | Q60823 Mouse | Q9WUA6 Mouse | P47196 Rat | P47197 Rat | Q63484 Rat
Images
HAK21112_1.jpg Fig1: Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (ET1609-51) at 1/2,000 dilution.

Lane 1: MCF7 cell lysate (20 µg/Lane)
Lane 2: A549 cell lysate (20 µg/Lane)
Lane 3: U-2 OS cell lysate (20 µg/Lane)
Lane 4: COS-1 cell lysate (20 µg/Lane)
Lane 5: NIH/3T3 cell lysate (20 µg/Lane)
Lane 6: RAW264.7 cell lysate (20 µg/Lane)
Lane 7: C6 cell lysate (20 µg/Lane)
Lane 8: PC-12 cell lysate (20 µg/Lane)
Lane 9: Mouse brain tissue lysate (20 µg/Lane)
Lane 10: Mouse heart tissue lysate (20 µg/Lane)
Lane 11: Mouse testis tissue lysate (20 µg/Lane)
Lane 12: Rat brain tissue lysate (20 µg/Lane)
Lane 13: Rat heart tissue lysate (20 µg/Lane)
Lane 14: Rat testis tissue lysate (20 µg/Lane)

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 24 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-51) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21112_2.jpg Fig2: Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/2,000 dilution and pan AKT antibody (HA721870) at 1/2,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate
Lane 3: SH-SY5Y cell lysate
Lane 4: SH-SY5Y treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 5: HEK-293 cell lysate
Lane 6: HEK-293 treated with 50μM LY294002 for 6 hours cell lysate
Lane 7: NIH/3T3 cell lysate
Lane 8: NIH/3T3 treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 9: C6 cell lysate
Lane 10: C6 treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 11: MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% BSA for 1 hour at room temperature. The primary antibody (HA722129) at 1/2,000 dilution and pan AKT antibody (HA721870) at 1/2,000 dilution were used in 5% BSA at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21112_3.jpg Fig3: Western blot analysis of Phospho-AKT (T308) on different lysates with Rabbit anti-Phospho-AKT (T308) antibody (HA722951) at 1/2,000 dilution.

Lane 1: Jurkat cell lysate
Lane 2: Jurkat treated with 100nM Calyculin A for 30 minutes cell lysate
Lane 3: HeLa cell lysate
Lane 4: HeLa treated with 100ng/mL Calyculin A for 30 minutes cell lysate
Lane 5: 293T cell lysate
Lane 6: 293T treated with 100nM Calyculin A for 15 minutes cell lysate

Lysates/proteins at 30 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722951) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21112_4.jpg Fig4: Immunocytochemistry analysis of MCF7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (ET1609-51) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (ET1609-51) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HAK21112_5.jpg Fig5: Immunofluorescence analysis of frozen mouse hippocampus tissue labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (ET1609-51).

The tissues were blocked in 3% BSA for 30 minutes at room temperature, washed with PBS, and then probed with the primary antibody (ET1609-51, green) at 1/100 dilution overnight at 4℃, washed with PBS. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was used as the secondary antibody at 1/200 dilution. Nuclei were counterstained with DAPI (blue). Image acquisition was performed with KFBIO KF-FL-400 Scanner.
HAK21112_6.jpg Fig6: Flow cytometric analysis of Jurkat cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (T308).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722951, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
HAK21112_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse spleen tissue untreated / treated with λpp / phospho-peptide / non-phospho-peptide with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA722129) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21112_8.jpg Fig8: Flow cytometric analysis of NIH/3T3 cells untreated (left) / treated with 100ng/mL PDGF for 1 hour (right) labeling Phospho-AKT (S473).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722129, 0.1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.