Invitrogen

AKT2 Monoclonal Antibody (4H7)

Advanced Verification

This Antibody was verified by KnockDown to ensure that the antibody binds to the antigen stated. View Details

View all (36) AKT2 antibodies

Datasheet

Datasheet

Product Image Gallery

AKT2 Antibody in Immunofluorescence (IF) — 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 (Product # MA1-034) 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.

AKT2 Antibody in Immunohistochemistry (IHC) — Immunohistochemistry was performed on biopsies of deparaffinized normal Human Medulla Oblongata and Human Esophageal cancer tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer for 20 minutes at 95°C. Following antigen retrieval tissues were blocked in 10% normal goat serum (Product # 31873) for 20 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Akt-2 (Product # MA1-034) overnight at 4°C in a humidified chamber. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate. Tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 400X magnification. Results demonstrate cytoplasmic localization of Akt-2.

AKT2 Antibody in Western Blot (WB) — Western blot analysis of Akt-2 was performed by loading 25 µg of lysate from Akt-2 SMART pool siRNA (Product # L-003001-00-0005) transfected or non-targeting control (Product # D-001810-10) transfected U2OS cells onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with Akt-2 mouse monoclonal antibody (Product # MA1-034) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Super Signal West Dura (Product # 34075).

AKT2 Antibody in Immunoprecipitation (IP) — Immunoprecipitation of Akt-2 was performed on HeLa cells. The antigen:antibody complex was formed by incubating 750 µg whole cell lysate with 2 µg of mouse monoclonal antibody recognizing Akt-2 (Product # MA1-034) overnight on a rocking platform at 4°C. The immune-complex was then captured on 50 µl Protein A/G Plus Agarose (Product # 20423). Captured immune-complexes were then washed extensively and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1%Tween for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing Akt-2 (Product # MA1-034) at a dilution of 1:1000 overnight rotating at 4°C. Membranes were washed in TBST and probed with Clean Blot IP Detection reagent (Product # 21230) at a dilution of 1:2000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Super Signal West Dura (Product # 34075).

AKT2 Antibody in ChIP assay (ChIP) — Chromatin immunoprecipitation analysis of 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 using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of an Akt2 monoclonal antibody (Product # MA1-034). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL 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. Data courtesy of the Innovators Program.

AKT2 Antibody in KnockDown — Antibody specificity was demonstrated by siRNA mediated knockdown of target protein. U2OS cells were transfected with AKT2 siRNA and decrease in signal intensity was observed in western blot application using Anti -AKT2 Monoclonal Antibody (Product # MA1-034). {KD}

Product Details

Tested Applications

Dilution

ChIP assay (ChIP)

1-3 µL

Immunocytochemistry (ICC)

1:10-1:100

Immunofluorescence (IF)

1:10-1:100

Immunohistochemistry (IHC)

1:200

Immunoprecipitation (IP)

2 µg

Western Blot (WB)

1:1000

Product Specifications

Species Reactivity

Human, Mouse, Non-human primate, Rat

Host / Isotype

Mouse / IgG1

Class

Monoclonal

Type

Antibody

Clone

4H7

Immunogen

Full length human recombinant protein of human AKT2 (NP_001617) produced in HEK293T cell.

Conjugate

Unconjugated

Form

Liquid

Concentration

1 mg/mL

Purification

Protein A

Storage buffer

PBS with 1mg/mL BSA, 30% glycerol

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_11153437

Target Information

This gene is a putative oncogene encoding a protein belonging to a subfamily of serine/threonine kinases containing SH2-like (Src homology 2-like) domains. The gene was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. Overexpression contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. The encoded protein is a general protein kinase capable of phophorylating several known proteins.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: AKT2 kinase; HIHGHH; murine thymoma viral (v-akt) homolog-2; murine thymoma viral (v-akt) oncogene homolog 2; PKB beta; PKBB; PKBBETA; PRKBB; Protein kinase Akt-2; Protein kinase B beta; protein kinase B, beta; putative v-akt murine thymoma viral oncoprotein 2; rac protein kinase beta; RAC protein kinase beta RAC-PK beta; RAC-BETA; RAC-beta serine/threonine protein kinase; RAC-beta serine/threonine-protein kinase; RAC-PK-beta; thymoma viral proto-oncogene 2; v-akt murine thymoma viral oncogene homolog 2

Gene Aliases: 2410016A19Rik; AKT2; AW554154; HIHGHH; PKB; PKBB; PKBBETA; PRKBB; RAC-BETA

UniProt ID: (Human) P31751, (Rat) P47197, (Mouse) Q60823

Entrez Gene ID: (Human) 208, (Rat) 25233, (Mouse) 11652

Molecular Function: annexin calcium-binding protein calmodulin intracellular calcium-sensing protein kinase non-receptor serine/threonine protein kinase protein kinase transfer/carrier protein transferase