All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 1/25000 dilution

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) treated with 100ng/ml PDGF for 1 hour whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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


Exposure time: 3 minutes

Blocking buffer and concentration: 5% NFDM/TBST

All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 1/1000 dilution

Lane 1 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates
Lane 2 : LNCaP (Human prostate carcinoma epithelial cell) treated with 0.1 uM Calyculin A for 30 minutes whole cell lysates
Lane 3 : A549 (Human lung carcinoma epithelial cell) whole cell lysates
Lane 4 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
Lane 6 : HUVEC (Human umbilical vein endothelial cell) whole cell lysates
Lane 7 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
Lane 8 : Mouse brain lysates
Lane 9 : Rat heart lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 56 kDa


Exposure time: 50 seconds

The basal expression level of AKT1 (phospho S473) varies in different cell lines reported by PMID: 19372546. But to detect clear signal, treatment is strongly recommended when using this antibody.

Blocking and diluting buffer: 5% NFDM/TBST

ab81283, at 1/100 dilution, staining AKT1 in untreated (left panel) and Phosphatase-treated (right panel) human cervical carcinoma by Immunohistochemistry using formalin-fixed, paraffin-embedded tissue

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 0.259 µg/ml

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) grown in serum free media overnight, whole cell lysate
Lane 2 : HeLa grown in serum free media overnight, then treated with 150nM Insulin for 5min, whole cell lysate
Lane 3 : HeLa grown in serum free media overnight, then treated with 150nM Insulin for 5min, whole cell lysate. Then the membrane was incubated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 56 kDa

Blocking and diluting buffer: 5% NFDM/TBST. 

Immunohistochemical analysis of Human HPV16 immortalized keratinocytes transfected with non-targeting siRNA, staining AKT1 (phospho S473) (green) with ab81283.
Antigen retrieval was performed by heat mediation in citrate buffer (pH 6). Samples were blocked with 10% goat serum before incubating with primary antibody (1/100). Fluoroscein-conjugated tyramide was used to detect staining.

MCF7 cells were incubated at 37°C for 2 hours with vehicle control (0 μM) and different concentrations of CCCP (ab 141229). Increased expression of AKT1 (phospho S473) (ab81283) in MCF7 cells correlates with an increase in CCCP concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab81283 at 2 μg/ml and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.

Dot blot analysis of AKT1 (phospho S473) phospho peptide (Lane 1) and AKT1 non-phospho peptide (Lane 2) labelling AKT1 (phospho S473) phospho peptide with ab81283 at a dilution of 1:1000 dilution (0.259μg/ml). A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1:20,000 dilution.

Blocking buffer: 5% NFDM/TBST. Dilution buffer: 5% NFDM /TBST.

All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 0.259 µg/ml

Lane 1 : LNCaP (human prostate carcinoma epithelial cell) whole cell lysate
Lane 2 : LNCaP treated with 100nM Cacyculin A for 30min whole cell lysate
Lane 3 : LNCaP treated with 100nM Cacyculin A for 30min whole cell lysate. Then the membrane was incubated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 56 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 1/1000 dilution

All lanes : HEK293 cell lysate

Predicted band size: 56 kDa

Insulin treatment: cells were starved overnight and then treated for 20 min (Insulin) at 100 ng/ml.

Phosphatase treatment: membrane strips were incubated with 200 ul of phosphatase (150 U/ml) at 37 degrees for 1 hour.

All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 1/1000 dilution

All lanes : NIH/3T3 cell lysate

Predicted band size: 56 kDa

PDGF treatment: cells were starved overnight and then treated for 1 h with PDGF at 100 ng/ml.

Phosphatase treatment: membrane strips were incubated with 200 ul of phosphatase (150 U/ml) at 37 degrees for 1 hour.

Primary : All Lanes : Anti AKT1 (phospho S473) antibody (ab81283) at 1:5000 dilution. Lane 1 = AKT1 (His tag) full length recombinant protein ab62279 - 50ng. Lane 2 = NIH3T3 serum starved overnight, 15ug. Lane 3 = NIH3T3 serum starved overnight and treated with PDGF-AB 50ng/mL for 1 hour, 15ug. Secondary : Lanes 1-3 : Goat polyclonal to Rabbit IgG H&L Pre-Adsorbed (HRP) at 1:5000 developed using the ECL technique. Performed under reducing conditions (50mM DTT, Sample heated at 60ºC). Predicted band size : 56kDa. Observed band size : 56kDa. Blocking step: 5% Milk in 50mM Tris+0.05% Tween for 1 hour at RT. Primary antibody buffer: 5% BSA in 50mM Tris+0.05% Tween overnight. Secondary antibody buffer: 5% Milk in 50mM Tris+0.05% Tween for 2 hours at RT. Exposure time : 5 minutes

NIH3T3 cells were starved overnight and treated with PDGF 50ng/mL or vehicle control for 1 hour prior to fixation with 4% paraformaldehyde. Levels of total Akt were measured using antibody ab81283 on an infrared in cell ELISA assay platform.

PC12 cells were incubated at 37°C for 24 hours with vehicle control (0 nM) and 1 μM of Galanin (1-15) (porcine, rat) (ab 141152). Decreased expression of AKT1 (phospho S473) (ab81283) in PC12 cells correlates with an increase in Galanin (1-15) (porcine, rat) concentration, as described in literature.

 Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 30μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated withab81283at 1 μg/ml andab8227at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.