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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
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.Species Reactivity:
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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
LI-COR is a registered trademark of LI-COR, Inc.
Odyssey is a registered trademark of LI-COR, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at firstname.lastname@example.org.