PhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA Kit (ab119654)

Overview

• Product namePhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA KitSee all AKT 1/2/3 (phospho Ser473) + total AKT 1/2/3 kits ...
• Tests
  1 x 96 well plate
• Sample type
  Cell culture extracts
• Assay typeSandwich
• Assay time
  1h 15m
• Species reactivity
  Reacts with: Mouse, Hamster, Human
  Predicted to work with: Rat
• Product overview

  PhosphoTracer assays use a traditional immuno-sandwich format, but with a major difference both the analyte and the assay reagents are added to the PhosphoTracer assay microplate at the same time. After a short incubation period, unbound assay reagents and analytes are washed away, and immuno-complexes containing both antibodies are detected. The process can take as little as 60 minutes to complete.

  PhosphoTracer kits also allow a higher degree of assay flexibility. In contrast to other ELISA formats, no antibodies are present on the assay microplate itself, so assays for several different targets can be performed in different wells on the same microplate. Simply mix the lysate with your target reagents of choice, using the microplate configuration of your choice.

  A whole new way of performing cellular assays, PhosphoTracer takes the hard work out of running a standard ELISA, while still giving the high quality results expected from a sandwich immunoassay. Fully self-contained kits are supplied in convenient 96-well packs. Simple to use and highly sensitive PhosphoTracer kits are designed to get results, fast.

  View the detection of AKT in HEK293, MCF7, HeLa and Jurkat cell lines.

   

  Abcam’s PhosphoTracer AKT 1/2/3 assay kits detect endogenous levels of AKT 1/2/3 (GenBank Accessions NP_001014431 [AKT1], NP_001617 [AKT2], and NP_859029 [AKT3]) in cellular lysates. Phospho-AKT 1/2/3 assays only detect AKT when phosphorylated at Ser473. Total AKT assays detect AKT regardless of phosphorylation state.
  
  
  
  The substrate used with the HRP conjugated detection antibody is a combination of 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) (wavelength exc/em = 530-540nm / 590-600nm), a highly sensitive and stable substrate for HRP) and ADHP Dilution Buffer (a stabilized H₂O₂ solution). Learn more about the fluorogenic substrate, ADHP.

• Notes

  Sensitivity: Phospho-AKT 1/2/3: 10 pg/ml (Tested with rhAKT1), AKT 1/2/3: 100 pg/ml (Tested with rhAKT1).

  Range: Phospho-AKT 1/2/3: 10-10,000 pg/ml, AKT 1/2/3: 100-100,000 pg/ml.

• Tested applicationsSandwich ELISA more details

Properties

• Storage instructionsStore at +4°C. Please refer to protocols.

• Components	1 x 96 tests
  96-well PhosphoTracer assay plate (stripwell)	1 unit
  Adherent plate seal	2 units
  ADHP (100X)	1 x 120µl
  ADHP Dilution Buffer	1 x 15ml
  Assay Control Lysate (lyophilized)	1 x 0.25ml
  Enhancer Solution	1 x 1ml
  Lysis Buffer (5X)	1 x 15ml
  Mouse monoclonal AKT 1/2/3 (24 assay points)	1 x 0.75ml
  Mouse monoclonal Phospho-AKT (Ser473) (72 assay points)	1 x 3ml
  Rabbit monoclonal Phospho-AKT 1/2/3 (HRP) (72 assay points)	3 x 0.75ml
  Rabbit polyclonal AKT 1/2/3 (HRP) (24 assay points)	1 x 0.75ml
  Stop Solution	1 x 2ml
  Wash Buffer (10X)	1 x 15ml

• Research Areas
  • Kits/ Lysates/ Other
  • Kits
  • ELISA Kits
  • ELISA Kits
  • Phosphoprotein ELISA kits

  • Signal Transduction
  • Protein Phosphorylation
  • Ser / Thr Kinases
  • Other Kinases

Applications

PhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA Kit images

• 

  Western blot - PhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA Kit (ab119654)
  Using Western blot, a significant increase in AKT 1/2/3 phosphorylation at Ser473 is detected in MCF7 cells treated with insulin for 15 minutes (+), compared with untreated cells (-), whereas no change in total AKT 1/2/3 levels was observed.
• 

  Sandwich ELISA - PhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA Kit (ab119654)
  Using the AKT 1/2/3 assay kits, a significant increase in AKT 1/2/3 phosphorylation at Ser473 is detected in MCF7 cells treated with insulin for 15 minutes (+), compared with untreated cells (-), whereas no change in total AKT 1/2/3 levels was observed.
• 

  Sandwich ELISA - PhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA Kit (ab119654)
  Using the PhosphoTracer phospho-AKT assay, the length of assay incubation time that was required to detect recombinant phospho-AKT was examined. After 15 mins, phospho-AKT concentrations of approximately 100 pg/mL were readily detected, while after 1 hour incubation, the limit of detection was less than 10 pg/mL. Longer incubation times had little effect on overall limit of detection that was observed.
• 

  Sandwich ELISA - PhosphoTracer AKT 1/2/3 (pS473) + total AKT 1/2/3 ELISA Kit (ab119654)
  Jurkat cells were harvested by centrifugation, resuspended in HBSS, and seeded at 10K/well (20 µL/well) in a rinsed PhosphoTracer assay plate. The cells were treated with various concentrations of wortmannin (10 µL/well) for 1 h at 37°C, then treated with 5% FCS (10 µL/well) for 15 min. The cells were lysed with 10 µL/well Lysis Mix Concentrate, with shaking for 10 min. Antibody Mix specific for phospho AKT (50 µL/well) was added to the lysate, and the plates were incubated for 1 hr at room temp, with shaking. Plates were washed and Substrate Mix was added. The plates were covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.

Protocols

Protocol Booklet