Recombinant Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283)

Blocking buffer and concentration: 5% NFDM/TBST

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.

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.

Blocking and diluting buffer: 5% NFDM/TBST.

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.

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

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.