Invitrogen

AKT2 Monoclonal Antibody (4H7)

Advanced Verification

This Antibody was verified by Knockdown to ensure that the antibody binds to the antigen stated.

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Datasheet

Protocols

Questions & Answers

Tech Support

Product Image Gallery

AKT2 Antibody in Immunocytochemistry (ICC/IF) — 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 (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 — 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

MA1-034

Applications	Tested Dilution	Publications
Western Blot (WB)	1:1,000	-
Immunohistochemistry (IHC)	1:200	-
Immunocytochemistry (ICC/IF)	1:10-1:100	-
Immunoprecipitation (IP)	2 µg	-
ChIP assay (ChIP)	1-3 µL	-

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. View antigen
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

Akt, protein kinase B (PKB), is a serine/threonine kinase, which is involved in many cellular signaling pathways and acts as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI 3-kinase). Akt2 is amplified and overexpressed in some human cancers.

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

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Bioinformatics

Protein Aliases: AKT2 kinase; murine thymoma viral (v-akt) homolog-2; murine thymoma viral (v-akt) oncogene homolog 2; PKB beta; 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 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: non-receptor serine/threonine protein kinase protein modifying enzyme