Anti-PRAS40 (phospho T246) antibody [RP23040013]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PRAS40 (phospho T246) antibody [RP23040013] (AB308042)

For immunofluorescence analysis, serum starved HeLa cells treated with 20 ng/mL PDGF for 15 minutes were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature for detection of PRAS40 (phospho T246) using ab308042 at 1/400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with DAPI F-actin (Panel c : red) was stained with Alexa Fluor® 594 Phalloidin Panel d is a merged image showing cytoplasmic localization Panel e shows competition with the PRAS40 (phospho T246) peptide The images were captured at 20X magnification

• WB

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Western blot - Anti-PRAS40 (phospho T246) antibody [RP23040013] (AB308042)

Western blot analysis of PRAS40 (phospho T246) was performed by loading 30 µg of untreated NIH/3T3 (lane 1) and NIH/3T3 treated with PDGF (100 ng/mL for 15 minutes) lysates (lane 2) using a 4-12% Bis-Tris gel, electrophoresis system and pre-stained protein standard. Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. PRAS40 (phospho T246) was detected at ~40 kDa using ab308042 at a 1 : 1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) (lane 3). Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody at a 1 : 5000 dilution and chemiluminescent detection was performed (ECL).

All lanes:

Western blot - Anti-PRAS40 (phospho T246) antibody [RP23040013] (ab308042) at 1/1000 dilution

Lane 1:

untreated NIH/3T3 lysate at 30 µg

Lane 2:

PDGF treated NIH/3T3 lysate at 30 µg

Lane 3:

Phosphopeptide and PDGF treated NIH/3T3 lysate at 30 µg

Secondary

All lanes:

HRP-conjugated Goat anti-Rabbit secondary antibody at 1/5000 dilution

Predicted band size: 27 kDa

Observed band size: 40 kDa

true

• WB

Supplier Data

Western blot - Anti-PRAS40 (phospho T246) antibody [RP23040013] (AB308042)

Western blot analysis of PRAS40 (phospho T246) in whole cell extracts of NIH-3T3 cells treated with 100 ng/mL PDGF for 15 min using ab308042 at a dilution of 2.5 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Results show a band at ~40 kDa.

All lanes:

Western blot - Anti-PRAS40 (phospho T246) antibody [RP23040013] (ab308042) at 100 ng/mL

Lane 1:

PDGF treated NIH-3T3 whole cell extract at 2.5 µg/mL

Lane 2:

Phosphopeptide and PDGF treated NIH-3T3 whole cell extract at 2.5 µg/mL

Predicted band size: 27 kDa

Observed band size: 40 kDa

false