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SimpleStep

AKT1 + AKT2 + AKT3 (pS473) ELISA试剂盒(ab176635)

Submit a review Q&A (1)References (3)
Typical Recombinant Protein Standard Curve
  • Typical cell lysate dilution series
  • Linearity of dilution
  • AKT (pS473) phosphorylation in response to insulin treatment.
  • Comparison of total AKT1 expression in different cell lines
  • Sandwich ELISA - AKT1 + AKT2 + AKT3 (pS473) ELISA Kit (ab176635)
  • Sandwich ELISA - AKT1 + AKT2 + AKT3 (pS473) ELISA Kit (ab176635)

Key features and details

  • One-wash 90 minute protocol
  • Sensitivity: 30 pg/ml
  • Range: 1 ng/ml - 100 ng/ml
  • Sample type: Cell Lysate, Tissue Homogenate
  • Detection method: Colorimetric
  • Assay type: Semi-quantitative
  • Reacts with: Mouse, Human

概述

  • 产品名称

    AKT1 + AKT2 + AKT3 (pS473) ELISA试剂盒
    参阅全部 AKT1 + AKT2 + AKT3 试剂盒

  • 检测方法

    Colorimetric

  • 精确度

    批次内
    样品 n Mean SD CV%
    HEK lysate 6 = 5.4%
    批次间
    样品 n Mean SD CV%
    HEK lysate 3 = 2.7%

  • 样品类型

    Cell Lysate, Tissue Homogenate

  • 检测类型

    Semi-quantitative

  • 灵敏度

    30 pg/ml

  • 范围

    1 ng/ml - 100 ng/ml

  • 回收率

    4.2 %

  • 检测时间

    1h 30m

  • 实验步骤

    One step assay

  • 种属反应性

    与反应: Mouse, Human
    预测可用于: Rat

  • 产品概述

    Abcam’s AKT 1/2/3 (pS473) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of AKT 1/2/3 (pS473) protein in Human and mouse cells.


    The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.


    As of October 2019, this kit was reformulated with new antibodies to maintain continued long term supply.

  • 说明

    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.

  • 经测试应用

    适用于: ELISAmore details

  • 平台

    Microplate

性能

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab176635于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
ELISA
Use at an assay dependent concentration.
说明
ELISA
Use at an assay dependent concentration.

图片

  • Typical Recombinant Protein Standard Curve
    Typical Recombinant Protein Standard Curve
  • Typical cell lysate dilution series
    Typical cell lysate dilution series

    Example of a typical AKT (pS473) cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.

  • Linearity of dilution
    Linearity of dilution

    Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1X Cell Extraction Buffer PTR. Data from duplicate measurements of AKT1 (pS473) are normalized and plotted.

  • AKT (pS473) phosphorylation in response to insulin treatment.
    AKT (pS473) phosphorylation in response to insulin treatment.

    Induction of AKT (pS473) phosphorylation in MCF-7 cells in response to insulin treatment. MCF-7 cells were cultured in 96-well tissue culture plates, serum-starved and treated (5 min) with a dose-range of insulin before cell lysis. Data from quadruplicate measurements of AKT (pS473) are plotted and compared against total AKT1 protein levels. Comparative AKT (pS473) and AKT1 (Total) data also shown by Western Blot.

  • Comparison of total AKT1 expression in different cell lines
    Comparison of total AKT1 expression in different cell lines

    Cell line analysis for total AKT1 from 20 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).

  • Sandwich ELISA - AKT1 + AKT2 + AKT3 (pS473) ELISA Kit (ab176635)
    Sandwich ELISA - AKT1 + AKT2 + AKT3 (pS473) ELISA Kit (ab176635)
    SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
  • Sandwich ELISA - AKT1 + AKT2 + AKT3 (pS473) ELISA Kit (ab176635)
    Sandwich ELISA - AKT1 + AKT2 + AKT3 (pS473) ELISA Kit (ab176635)
    To learn more about the advantages of SimpleStep ELISA® kits see here.

文献 (3)

发表研究结果有使用 ab176635？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab176635 被引用在 3 文献中.

  • Ahmed HMS  et al. Acute and chronic metabolic effects of carvedilol in high-fructose, high-fat diet-fed mice: implication of β-arrestin2 pathway. Can J Physiol Pharmacol 100:68-77 (2022). PubMed: 34570983
  • El-Fayoumi S  et al. Pioglitazone Enhances β-Arrestin2 Signaling and Ameliorates Insulin Resistance in Classical Insulin Target Tissues. Pharmacology 106:409-417 (2021). PubMed: 34082428
  • Wang P  et al. Potential Involvement of Adiponectin Signaling in Regulating Physical Exercise-Elicited Hippocampal Neurogenesis and Dendritic Morphology in Stressed Mice. Front Cell Neurosci 14:189 (2020). PubMed: 32774242

客户评价及客户问答

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Question

Hi, I have noticed that in the protocol for the SimpleSet ELISA kit for p53 in the step: 11.2 Preparation of extracts from adherent cells by direct lysis You recommend solubilizing the cells by addition of chilled 1X Cell Extraction Buffer PTR directly to a 15 cm diameter plate. Would I be able to scale the volume of lysis buffer down to a 96 well plate format? This could then be transfered into the 96 well ELISA plate directly? Also why is there a need to scrape the cells afterwards? I have noticed that in kits for other targets (such as pAKT473) this step is not necessary. Can this kit be used with cell lysates (similarly to other kits such as the pAKT473)?

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Answer



Similarly to the AKT protocol, for the p53 kit, 96WP samples can be processed. If this is the case, 0.1mL is an appropriate starting volume for lysing cells in a microplate. Extracts should be pipetted up/down several times to fully solubilize.

The main caution to take is to make certain that you can achieve a workable concentration of extract from the 96WP. Please have a look at Figures 2 &3 in the p53 Simple Step ELISA booklet (ab171571, page 20) as guidance for concentrations to be within the working range of the assay for some example cell lines.

I would recommend running a pilot experiment to determine what concentrations can be achieved for your given cell line and culture conditions – the results should then dictate the lysis volume and if the extract needs to be further diluted in extraction buffer before adding to the ELISA plate.

We think scraping increases the recovery of extract on large plates but it is not strictly necessary (and of course is impossible in 96WP).

It is also advisable to take care in washing cells in PBS if the treatment or culture conditions have caused any cells to become loosely attached. You may also want to consider normalizing extract loads to the ELISA plate (e.g. by BCA assay) if the treatments are causing an effect on cell number. Note that 50uL of sample always needs to be added to the SSE plate.

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