Western blot analysis of extracts from MKN-45 cells, untreated or treated with c-Met kinase inhibitor SU11274 (1 µM, 2 hr), using Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb. Western blot image was obtained using the Odyssey® Infrared Imaging System (LI-COR® Biotechnology).
Western blot analysis of extracts from A-431 cells, untreated or treated with Human Epidermal Growth Factor (hEGF) #8916 (10 ng/ml, 20 min), using Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb (upper), or Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (lower). Western blot images were obtained using the Odyssey® Infrared Imaging System (LI-COR® Biotechnology).
|Peptide ELISA (DELFIA)||1:1000|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
(DELFIA® is a registered trademark of PerkinElmer, Inc.)
posted June 2005
revised September 2007
Protocol Id: 34
Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb recognizes endogenous proteins containing phospho-Ser/Thr preceded by Arg at positions -5 and -3 in a manner largely independent of the surrounding amino acid sequence. Minor cross-reactivity is observed for proteins that contain phospho-Ser/Thr preceded by Arg at position -3 only. No cross-reactivity is observed with the corresponding nonphosphorylated sequences or with other phospho-Ser/Thr-containing motifs.
All Species Expected
Monoclonal antibody is produced by immunizing animals with an Akt substrate peptide library.
An important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (1,2). Akt plays a central role in mediating critical cellular responses including cell growth and survival, angiogenesis, and transcriptional regulation (3-5). While a number of Akt substrates are known (such as GSK-3, Bad, and caspase-9) many important substrates await discovery. Akt phosphorylates substrates only at Ser/Thr in a conserved motif characterized by Arg at positions -5 and -3 (6). Phospho-Akt substrate-specific antibodies from Cell Signaling Technology are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.