Phospho- & total AKT 1/2/3, Phospho- & total pan AKT Cellular Assay Kits

Phospho-AKT 1/2/3 (Ser473), Phospho-pan AKT (Ser473), Phospho pan AKT (Thr308), total AKT 1/2/3 & total pan AKT assays for oncology and metabolism studies

Also known as protein kinase B (PKB), Akt is an oncogene playing a key role in controlling apoptosis, cell proliferation, transcription, cell migration and glucose metabolism.

Cisbio has developed specific assays for Akt isoforms: Akt 1 involved in cell survival, Akt2 involved in insulin pathway, Akt3 involved in neurosciences as well as a pan Akt assay to monitor all isoforms.*

Our offer include:

  • 4 assays to detect Ser473 phosphorylation of Akt1, Akt2, Akt3 and pan Akt
  • 1 specific assay to detect Thr308 phosphorylation of all isoforms (pan Akt)
  • 4 Total Akt assays to normalize your results versus steady state expression of Akt1, Akt2, Akt3 or all isoforms (pan Akt)

 

Available assays Ser 473 Thr 308 Total
Pan Akt done done done
Akt1 done - done
Akt2 done - done
Akt3 done - done

 

   Check our lysis buffer compatibility

Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total pAkt assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-Akt antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with pAkt, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-Akt can be quantitatively detected using the HTRF phospho-Akt and total p53 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Product performances

1. PHOSPHO-AKT (THR308) ASSAY IGF DOSE-RESPONSE ON CHO-M1 CELLS

Results obtained on CHO cells plated using 2 cell densities (25,000 - 50,000 cells per well). After being starved for 24 hours, cells were stimulated for 10 minutes with various concentrations of IGF. Phosphorylated AKT on Thr308 was measured after 3.5 hours using the two-plate assay protocol.

2. PHOSPHO-AKT (SER473) ASSAY IGF DOSE-RESPONSE ON HEK 293 CELLS

Results obtained on HEK cells plated using 3 cell densities (12,500 - 25,000 - 50,000 cells per well). After being starved for 24 hours, cells were stimulated for 10 minutes with various concentrations of IGF. Phosphorylated AKT was measured after 3.5 hours using the two-plate assay protocol.

3. TOTAL-AKT ASSAY USED TO CONTROL THE PHOSPHORYLATION STATUS OF AKT

 

HEK293 cells (100,000 cells/well) were activated with IGF-1 for 10 min, using the two-plate assay protocol of the Phospho-AKT(Ser473) and Total-AKT assays.

As expected, results obtained show a dose-response increase of AKT phosphorylation upon IGF-1 stimulation, while the AKT expression level remains weakly constant.

4. Phospho- & total AKT1, AKT2, AKT3 assays: Inhibitory effect of blocking peptide

SH-SY5Y cells were stimulated with 1 µM of human IGF-1 for 15 min at 37°C – 5% CO2. After stimulation, media was removed and cells were lysed with lysis buffer 1X for 30 min at RT under gentle shaking. 14 µL of lysates were transferred into 384-well sv white microplates and 2 µL of different concentration of blocking peptide specific for AKT1, AKT2 or AKT3 was added before adding 4 µL of the HTRF phospho AKT1 (Ser473), phospho-AKT2 (Ser473), phospho-AKT3 (Ser473), total AKT1, total AKT2 or total AKT3 detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

Simplified Pathway

AKT (or protein kinase B) plays a key role in controlling survival and apoptosis. This serine/threonine protein kinase is regulated by insulin and various growth and survival factors, to work in a wortmannin-sensitive pathway involving PI 3 kinase. When the Pleckstrin Homology (PH) domain of AKT binds to phosphoinositides, AKT can be phosphorylated by two different kinases, PDK1 at threonine 308 and mTORC2 (mammalian target of rapamycin) at serine 473, which switch on AKT activation. Besides being a downstream effector of PI3-kinases, AKT may also be activated in a PI 3-kinase-independent manner.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
AKT1 phospho S473 kit - 500 tests63ADK078PEG
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AKT1 phospho S473 kit - 10,000 tests63ADK078PEH
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AKT1 total kit - 500 tests63ADK079PEG
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AKT1 total kit - 10,000 tests63ADK079PEH
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AKT2 phospho S473 kit - 500 tests63ADK080PEG
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AKT2 phospho S473 kit - 10,000 tests63ADK080PEH
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AKT2 total kit - 500 tests63ADK081PEG
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AKT2 total kit - 10,000 tests63ADK081PEH
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AKT3 phospho S473 kit - 500 tests63ADK082PEG
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AKT3 phospho S473 kit - 10,000 tests63ADK082PEH
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AKT3 total kit - 500 tests63ADK083PEG
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AKT3 total kit - 10,000 tests63ADK083PEH
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AKT phospho-S473 kit - 500 tests64AKSPEG
AKT phospho-S473 kit - 10,000 tests64AKSPEH
AKT phospho-S473 kit -1 x 96 tests64AKSPET
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AKT phospho-T308 kit - 500 tests64AKTPEG
AKT phospho-T308 kit - 10,000 tests64AKTPEH
AKT total kit - 500 tests64NKTPEG
AKT total kit - 10,000 tests64NKTPEH
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AKT total kit - 1 x 96 tests64NKTPET
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Companion products

DescriptionCat. noProduct insertMSDS
AKT phospho-S473 kit control lysate62AKSTDA
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AKT phospho-T308 kit control lysate62AKTTDA
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AKT1 phospho S473 control lysate63ADK078TDA
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AKT1 total control lysate63ADK079TDA
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AKT2 phospho S473 control lysate63ADK080TDA
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AKT2 total control lysate63ADK081TDA
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AKT3 phospho S473 control lysate63ADK082TDA
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AKT3 total control lysate63ADK083TDA
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Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
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AKT total kit control lysate64NKTTDA
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