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

Reviews (1)Q&A (6)References (5)

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.

  • Platform

    Microplate

Properties

Associated products

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

References

This product has been referenced in:

  • 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). Read more (PubMed: 30905432) »
  • Riddle MR  et al. Insulin resistance in cavefish as an adaptation to a nutrient-limited environment. Nature 555:647-651 (2018). Read more (PubMed: 29562229) »
See all 5 Publications for this product

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

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

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 ?

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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

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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.

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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.

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Compatibility with Porcine cell (LLC-PK1)

 Average Good 4/5 (Ease of Use)
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.

Abcam user community

Verified customer

Submitted Sep 18 2013

Question

KIndly contacted us to inquire about alternative lysis buffers.

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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.

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Question

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

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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.

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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,

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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.

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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.

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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.

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