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    akt-ps473--total-akt-elisa-kit-ab126433.pdf

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Akt (pS473) + total Akt ELISA Kit (ab126433)

  • Datasheet
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  • Protocol Booklet
Reviews (1)Q&A (6)References (8)

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Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
  • Western blot - Akt (pS473) + total Akt ELISA Kit (ab126433)
  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)

Key features and details

  • Sample type: Cell Lysate
  • Detection method: Colorimetric
  • Assay type: Semi-quantitative
  • Reacts with: Mouse, Rat, Human

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Overview

  • Product name

    Akt (pS473) + total Akt ELISA Kit
  • Detection method

    Colorimetric
  • Sample type

    Cell Lysate
  • Assay type

    Semi-quantitative
  • Assay time

    5h 00m
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    ab126433 is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cell lysates. By determining phosphorylated Akt protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blot analysis. 


    This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of phospho-Akt (Ser473) and total Akt in human, mouse and rat cell lysates (help normalize the results of phospho-Akt from different cell lysate being compared). A pan Akt antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and Akt present in a sample is bound to the wells by the immobilized antibody. The wells are washed and anti-phospho-Akt (Ser473) or anti-pan-Akt is used to detect phosphorylated or total Akt. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Akt (Ser473) or total Akt bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.


    Get higher sensitivity in only 90 minutes with AKT 1/2/3 pS473 + AKT1 ELISA Kit (ab176657) from our SimpleStep ELISA® range.

  • Notes

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • Platform

    Microplate

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    500X HRP-conjugated anti-rabbit IgG 1 x 25µl
    20X Wash Buffer 1 x 25ml
    2X Cell Lysis Buffer 1 x 5ml
    5X Assay Diluent 1 x 15ml
    Akt Microplate (12 strips x 8 wells) coated with anti-pan Akt antibody 1 unit
    Detection Antibody Akt (Ser473): rabbit anti-phospho-Akt (Ser473) 1 vial
    Detection Antibody Akt: rabbit anti-pan-Akt 1 vial
    Positive Control: lyophilized NIH3T3 cell lysate 1 vial
    Stop Solution 1 x 8ml
    TMB One-Step Substrate Reagent 1 x 12ml
  • Research areas

    • Cell Biology
    • Cell Cycle
    • Kinases/Phosphatases
    • Other
    • Neuroscience
    • Neurotransmission
    • Intracellular Signaling
    • Kinases
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • MAPK Pathway
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • PKB / AKT
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases
    • Signal Transduction
    • Growth Factors/Hormones
    • Insulin / Insulin-like
    • Signal Transduction
    • Protein Trafficking
    • Vesicle Transport
    • Regulation
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • HLH / Leucine Zipper
    • HLH / Leucine Zipper
    • Epigenetics and Nuclear Signaling
    • Cell cycle
    • Apoptosis
    • Nuclear
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Transcription Factors
    • Cancer
    • Cell cycle
    • Kinases/phosphatases
    • Other
    • Cancer
    • Signal transduction
    • Protein phosphorylation
    • Serine/threonine kinases
    • AKT
    • Kits/ Lysates/ Other
    • Kits
    • ELISA Kits
    • ELISA Kits
    • Signal transduction proteins ELISA kits
    • Kits/ Lysates/ Other
    • Kits
    • ELISA Kits
    • ELISA Kits
    • Phosphoprotein ELISA kits
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Apoptosis
    • Cancer
    • Cell Death
    • Apoptosis
    • Metabolism
  • Cellular localization

    AKT1/2/3: Cytoplasm. Membrane. Membrane-associated after cell stimulation leading to its translocation. AKT1 (total): Cytoplasm. Nucleus. Cell membrane. Nucleus after activation by integrin-linked protein kinase 1 (ILK1). Nuclear translocation is enhanced by interaction with TCL1A. Phosphorylation on Tyr-176 by TNK2 results in its localization to the cell membrane where it is targeted for further phosphorylations on Thr-308 and Ser-473 leading to its activation and the activated form translocates to the nucleus.
  • Alternative names

    • AKT
    • AKT 1
    • AKT1
    • AKT1_HUMAN
    • Akt2
    • AKT2_HUMAN
    • Akt3
    • AKT3 kinase
    • AKT3_HUMAN
    • C AKT
    • cAKT
    • DKFZp434N0250
    • HIHGHH
    • MGC99656
    • MPPH
    • Murine thymoma viral (v-akt) homolog 2
    • murine thymoma viral (v-akt) homolog-2
    • Oncogene AKT2 protein kinase B beta
    • PKB
    • PKB alpha
    • PKB beta
    • PKB gamma
    • PKB-ALPHA
    • PKBB
    • PKBBETA
    • PKBG
    • PRKBA
    • PRKBB
    • PRKBG
    • Protein kinase Akt 2
    • Protein kinase Akt-2
    • Protein kinase Akt-3
    • Protein Kinase AKT3
    • Protein kinase B
    • Protein Kinase B Alpha
    • Protein kinase B beta
    • Protein kinase B gamma
    • Proto-oncogene c-Akt
    • RAC
    • RAC Alpha
    • RAC beta
    • RAC gamma
    • RAC gamma serine/threonine protein kinase
    • Rac protein kinase alpha
    • rac protein kinase beta
    • RAC Serine/Threonine Protein Kinase
    • RAC-alpha serine/threonine-protein kinase
    • RAC-BETA
    • RAC-beta serine/threonine-protein kinase
    • RAC-gamma serine/threonine-protein kinase
    • RAC-PK-alpha
    • RAC-PK-beta
    • RAC-PK-gamma
    • RACbeta
    • RACPK Gamma
    • Serine threonine protein kinase Akt 3
    • Serine threonine protein kinase Akt3
    • STK 2
    • STK-2
    • STK2
    • v akt murine thymoma viral oncogene homolog 1
    • v akt murine thymoma viral oncogene homolog 2
    • V akt murine thymoma viral oncogene homolog 3
    • V akt murine thymoma viral oncogene homolog 3 (protein kinase B, gamma)
    • V akt murine thymoma viral oncogene homolog 3 protein kinase B gamma
    • V-AKT murine thymoma viral oncogene homolog 2
    • vAKT Murine Thymoma Viral Oncogene Homolog 1
    see all
  • Database links

    • Entrez Gene: 10000 Human
    • Entrez Gene: 10000 Human
    • Entrez Gene: 1385 Human
    • Entrez Gene: 207 Human
    • Entrez Gene: 208 Human
    • Entrez Gene: 5594 Human
    • Entrez Gene: 5595 Human
    • Entrez Gene: 11651 Mouse
    • Entrez Gene: 11652 Mouse
    • Entrez Gene: 12912 Mouse
    • Entrez Gene: 23797 Mouse
    • Entrez Gene: 23797 Mouse
    • Entrez Gene: 26413 Mouse
    • Entrez Gene: 26417 Mouse
    • Entrez Gene: 16590 Rat
    • Entrez Gene: 24185 Rat
    • Entrez Gene: 25233 Rat
    • Entrez Gene: 29414 Rat
    • Entrez Gene: 29414 Rat
    • Entrez Gene: 50689 Rat
    • Entrez Gene: 81646 Rat
    • Omim: 123810 Human
    • Omim: 164730 Human
    • Omim: 164731 Human
    • Omim: 176948 Human
    • Omim: 601795 Human
    • Omim: 611223 Human
    • SwissProt: P16220 Human
    • SwissProt: P27361 Human
    • SwissProt: P28482 Human
    • SwissProt: P31749 Human
    • SwissProt: P31751 Human
    • SwissProt: P31751 Human
    • SwissProt: Q9Y243 Human
    • SwissProt: Q9Y243 Human
    • SwissProt: P31750 Mouse
    • SwissProt: P63085 Mouse
    • SwissProt: Q01147 Mouse
    • SwissProt: Q60823 Mouse
    • SwissProt: Q63844 Mouse
    • SwissProt: Q9WUA6 Mouse
    • SwissProt: Q9WUA6 Mouse
    • SwissProt: P15337 Rat
    • SwissProt: P21708 Rat
    • SwissProt: P47196 Rat
    • SwissProt: P47197 Rat
    • SwissProt: P63086 Rat
    • SwissProt: Q63484 Rat
    • SwissProt: Q63484 Rat
    • Unigene: 431850 Human
    • Unigene: 498292 Human
    • Unigene: 516646 Human
    • Unigene: 631535 Human
    • Unigene: 235194 Mouse
    • Unigene: 10506 Rat
    see all

Images

  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    MCF7 cells were stimulated for five minutes with 1 ug x mL-1 of insulin (ab123768). Cell lysates were assessed for total Akt and Akt(pS473), shown as OD (450 nm) after background signal was subtracted (duplicates +/- SD).
  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    The NIH3T3 cells were treated with recombinant human PDGF for 10 minutes to induce phosphorylation of Akt. Serial dilutions of lysates were analyzed in this ELISA.
  • Western blot - Akt (pS473) + total Akt ELISA Kit (ab126433)
    Western blot - Akt (pS473) + total Akt ELISA Kit (ab126433)
    NIH3T3 cells were treated or untreated with recombinant human PDGF for 10 min. Cell lysates were analyzed by Western Blot.
  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    NIH3T3 cells were treated or untreated with recombinant human PDGF for 10 min. Cell lysates were analyzed using this phosphoELISA.
  • Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    Sandwich ELISA - Akt (pS473) + total Akt ELISA Kit (ab126433)
    A431 cells were treated with recombinant human EGF at 37°C for 20 min. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (8)

Publishing research using ab126433? Please let us know so that we can cite the reference in this datasheet.

ab126433 has been referenced in 8 publications.

  • Tajima Y  et al. Association of surfactant protein D with pulmonary metastases from colon cancer. Oncol Lett 20:322 (2020). PubMed: 33123238
  • Hu S  et al. Panax notoginseng saponins suppress lipopolysaccharide-induced barrier disruption and monocyte adhesion on bEnd.3 cells via the opposite modulation of Nrf2 antioxidant and NF-?B inflammatory pathways. Phytother Res 33:3163-3176 (2019). PubMed: 31468630
  • Lin Q  et al. Exosome-like nanoplatform modified with targeting ligand improves anti-cancer and anti-inflammation effects of imperialine. J Control Release 311-312:104-116 (2019). PubMed: 31484040
  • Guan Y  et al. Chloride channel-3 is required for efficient tumour cell migration and invasion in human cervical squamous cell carcinoma. Gynecol Oncol 153:661-669 (2019). PubMed: 30905432
  • Riddle MR  et al. Insulin resistance in cavefish as an adaptation to a nutrient-limited environment. Nature 555:647-651 (2018). PubMed: 29562229
  • Ding WZ  et al. MicroRNA-497 regulates cell proliferation in hepatocellular carcinoma. Oncol Lett 11:1081-1088 (2016). WB . PubMed: 26893696
  • Gerdes JM  et al. Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents. Nat Commun 5:5308 (2014). Sandwich ELISA ; Mouse . PubMed: 25374274
  • Arroba AI  et al. Loss of protein tyrosine phosphatase 1B increases IGF-I receptor tyrosine phosphorylation but does not rescue retinal defects in IRS2-deficient mice. Invest Ophthalmol Vis Sci 54:4215-25 (2013). PubMed: 23702782

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

Compatibility with Porcine cell (LLC-PK1)

Average Good 4/5 (Ease of Use)
Abreviews
Abreviews
I have used this product to measure the increase in the phosphorylation of AKT when treated with TGF-beta. I found the values to be consistent across the control and TGF-beta treated groups. The phosphorylation of AKT also increased on treatment with TGF-beta as the literature suggests. I feel this product to be reliable in the porcine model as well.
However, the problem I had with the kit was with the standard curve. Firstly, the lyophillized cell lysate that was provided precipitated and I could not obtain a proper standard curve. Moreover, I feel that it would be a good idea for abcam to quantitate the amount of AKT present in the cell lysate they provide so that the results may be expressed as (amount of pAKT/ amount of total AKT) instead of the (absorbance of pAKT/ absorbance of total AKT) as it is being done now.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Sep 18 2013

Question

Recently, I am running an competition sandwich ELISA experiment , and my ligands that I am using react in the presence of Zn2+. Unfortunately, the assay diluent buffer and the washing buffer of the ELISA kit that I ordered are PBS buffers which will cause precipitation of zinc ions or decrease the concentration of it by forming complexes. I would like to ask if there is any TRIS buffer that I could use instead, as a diluent and washing buffer for the ab126433 ELISA kit ?

Read More

Abcam community

Verified customer

Asked on Aug 27 2014

Answer


We haven’t directly used TRIS based buffers with ab126433 Akt (pS473) + total Akt ELISA Kit, so I am sorry we cannot confirm for sure this type of alteration will work. However, you can certainly try, and keep us informed if there are any issues.
For your information, the diluent buffer is a proteinacious buffer with detergent, and the buffer is a detergent based buffer

Read More

Sam Washer

Abcam Scientific Support

Answered on Aug 27 2014

Question

I want the exact amount/volume (in mls or microliters) of protease and phosphatase inhibitor added to lysate buffer please. (I will use protease inhibitor cocktail and NaF,Beta glycerophosphate and sodium orthovanadate as phosphatase inhibitors) I need concentrations and volumes of the phosphatase inhibitors chemicals.

Read More

Abcam community

Verified customer

Asked on Mar 05 2014

Answer


Here is a link to our guide for use of protease and phosphatase inhibitors:
https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A2
For phosphatase inhibitors, recommended NaF and Na3VO4 working concentrations are 1mM.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Mar 05 2014

Question

KIndly contacted us to inquire about alternative lysis buffers.

Read More

Abcam community

Verified customer

Asked on Aug 01 2013

Answer

For best results, the we recommend use of the lysis buffer included with the kit to prepare lysate samples. However, other similar lysis buffers, like RIPA, will likely also work. If using a different lysis buffer than the one that is supplied in the kit, please be sure the buffer meets the following guidelines: A) has relatively low salt content (700 mM or less) B) does not contain sodium azide C) does not contain >0.1% SDS D) does not contain >10 mM reducing agents (beta-mercaptoethanol or dithiothreitol) This would include any buffers used for immunoprecipitations, including RIPA buffer.

Read More

Keith B

Abcam Scientific Support

Answered on Aug 01 2013

Question

Please clarify if ab126433 measures both Akt (pS473) and total Akt simultaneously in the same well?

Read More

Abcam community

Verified customer

Asked on Feb 18 2013

Answer


I can confirm that this kit uses a plate coated with pan Akt antibody. After samples have been added, either anti-phospho-Akt (Ser473) or anti-pan-Akt antibody is used to detect phosphorylated or total Akt. This means that either Akt (pS473) or total Akt is detected in one well. I would suggest running 2 identical samples in parallel and then detecting Akt (pS473) in one and total Akt in the other so that you may get both readings for each sample.

Read More

Abcam Scientific Support

Answered on Feb 18 2013

Question

we would be interested in the pAkt ELISA Kit ab126433, that is out of
stock. Is it still possible to order?
I have got another question regarding quantitative analysis of
extracellular matrix components of tissue fragments. Which method
could you recommend?
Thank you very much for your help,
best regards,

Read More

Abcam community

Verified customer

Asked on Jun 27 2012

Answer

Thank you for contacting us.


I am happy to confirm that ab126433 is in stock in our US office and if you order it today before 4pm you should receive it on the 3rd of July.


We are happy to help customers find the most suitable method for their research purposes; however we are not specialized in this particular field and do not want to recommend a method that may be unsuitable. We encourage customers to consult the latest literature available through PubMed and other resources in order to find the most up-to-date information about their specific research interests.

Depending on the target you are interested I can only recommend standard methods like IHC or WB. When I google "analysis of extracellular matrix components of tissue fragments" I get many publications that might be of help when deciding what the optimal way for analysis is.

I am sorry that I could not be more helpful, but I hope that the available literature in this area can provide some clarification. Please do not hesitate to contact us again with other needs or with any questions about our products.

Read More

Abcam Scientific Support

Answered on Jun 27 2012

Question

Product code: 126433
Inquiry: The customer wants to buy the Akt (pS473) + total Akt ELISA Kit (ab126433) .But the kits is out of stock .He wants to know when he can get the kits. Would you please help him provide the information? Thank you very much.

Read More

Abcam community

Verified customer

Asked on Jun 21 2012

Answer

Thank you for contacting us.

We have just received a shipment into stock (this informationwill be updated on the website shortly) and will be receiving more in the future. If your customer were to place an order we would be able to send them out as usual to you.

Many thanks for your help.

Read More

Abcam Scientific Support

Answered on Jun 21 2012

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