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

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

NIH3T3 cells starved overnight and treated with PDGF 50ng/mL for 1 hour (A) or vehicle (B). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton-X100. Primary antibody ab81283 was used at 1:4000 dilution and secondary antibody Dylight GAR594 (ab96897) at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST. 

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 withab81283at 2 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.

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.

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.

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.

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.

ab81283 staining AKT1 (phospho S473) in Human peritoneal tumor tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/500) for 2 hours. An Alexa Fluor^® 647-conjugated Donkey anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.

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