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    tmb-elisa-substrate-slowest-kinetic-rate-ab171527.pdf

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TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)

  • Datasheet
  • SDS
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ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
  • ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
  • ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)

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Overview

  • Product name

    TMB ELISA Substrate (Slowest Kinetic Rate)
    See all ELISA substrate reagents
  • Tested applications

    Suitable for: ELISAmore details
    Unsuitable for: IHC-Fr or IHC-P
  • General notes

    Abcam’s TMB ELISA Substrate (Slowest Kinetic Rate) contains 3,3’,5,5’-tetramethylbenzidine in a mildly acidic buffer. The substrate is supplied as a ready to use solution. Unreacted substrate should be colorless or very light yellow in appearance. When this substrate system is reacted with peroxidase, a soluble blue reaction product is obtained. The reaction can be stopped using appropriate stop solution (see below), producing a soluble yellow or soluble blue reaction product, depending upon the stop reagent used, which is stable for at least 1 hour. ab171527 is not recommended for membrane or immunohistochemical applications that require a precipitating reaction product.

    Product Use:
    1. Allow substrate to equilibrate to room temperature (25°C) before use. Be sure to protect it from light during this process.
    2. After final antibody incubation and wash steps, tap the ELISA plate to remove excess liquid and add 100 µL of TMB substrate. The amount of substrate used should be tailored to your assay requirements.
    3. A soluble, blue color will slowly develop and can be read at 650 nm.
    4. Color development time can range between 10-40 minutes depending on the assay and will also depend on the method used to stop the reaction (see below). It is recommended that development time be optimized for your particular assay condition, however, a substrate color development time of less than 10 minutes is not recommended to ensure consistency of results.
    5. In endpoint assays, the substrate reaction can be stopped using equal volumes of 1 N HCl, 0.6 N sulfuric acid, or one of the Stop Solutions for TMB Substrates. (See 6a and 6b for O.D. recommendations)
    6. Stop Solutions have been optimized based on the desired endpoint.
    a) The 450 nm Stop Solution is ab171529. They produce a soluble yellow product. Since stopping the reaction increases sample absorbance values approximately 2.5 fold, the maximum O.D. at 650 nm should be 1.2 to 1.3.
    b) The 650 nm Stop Solution is ab171531, which maintains the soluble blue color. Stopping with these reagents allows for the generation of O.D. values at 650 nm of up to 3.0, above which absorbance values are no longer linear with concentration. The 650 nm Stop Reagents are recommended for assays that need a large dynamic range without sacrificing detection limit.
    7. If the reaction color is too intense, it is recommended that you dilute either your antibody or conjugate. Dilution of the TMB substrate is NOT recommended.

    Storage Instructions:

    Exposure to direct sunlight and other UV sources should be avoided due to the light sensitive nature of the TMB molecule.

Properties

  • Form

    Liquid
  • Storage instructions

    Store at +4°C. Please see notes section.
  • Storage buffer

    pH: 1.00
    Constituents: 0.05% 3,3',5,5'-Tetramethylbenzidine, 79% Water, 0.1% Hydrogen peroxide
  • Concentration information loading...

Associated products

  • Related Products

    • TMB ELISA Substrate (High Sensitivity) (ab171523)
    • TMB ELISA Substrate (Fast Kinetic Rate) (ab171524)
    • TMB ELISA Substrate (Slow Kinetic Rate) (ab171525)
    • TMB ELISA Substrate (Slower Kinetic Rate) (ab171526)
    • 450 nm Stop Solution for TMB Substrate (ab171529)
    • 650 nm Stop Solution for TMB Substrate (ab171531)
    • Phosphate Buffered Saline (PBS) Casein ELISA Reagent (ab171532)
    • Tris Buffered Saline (TBS) Casein ELISA Reagent (ab171533)
    • Immunoassay Blocking Buffer (ab171534)
    • Immunoassay Blocking (BSA Free) (ab171535)
    • AP Stability Conjugate Reagent (ab171536)
    • HRP Stability Conjugate Reagent (ab171537)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab171527 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ELISA
1/1. 100 µL of substrate solution is added to each well.
Notes
ELISA
1/1. 100 µL of substrate solution is added to each well.
Application notes
Is unsuitable for IHC-Fr or IHC-P.

Images

  • ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
    ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
    Comparison of the kinetic curves of several TMB substrates at 500 pg/mL msIgG monitored over time at 650 nm.
  • ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
    ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
    Expanded graph at the lower concentrations using TMB ELISA Substrate (Fast Kinetic Rate) (ab171524) and TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527). Both ab171524 and ab171527 have the same detection limit.
  • ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
    ELISA - TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527)
    Standard curve comparison for TMB ELISA Substrate (Fast Kinetic Rate) (ab171524) and TMB ELISA Substrate (Slowest Kinetic Rate) (ab171527) stopped and read at 450 nm. Results show an extended dynamic range when ab171527 is used.

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab171527? Please let us know so that we can cite the reference in this datasheet.

ab171527 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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Question


1-) What are the main differences between ab171524 (TMB ELISA Substrate - Fast Kinetic Rate) and ab171527 (TMB ELISA Substrate - Slowest Kinetic Rate)

2-) When should I choose using ab171524 or ab171527? I mean, is there any special situation (or condition) in which I should prefer one of these kit instead of the other?

Read More

Abcam community

Verified customer

Asked on Aug 21 2013

Answer



ab171524 (TMB ELISA Substrate - Fast Kinetic Rate) and ab171527 (TMB ELISA Substrate - Slowest Kinetic Rate) differ in the rate of color development and sensitivity.

TMB ELISA Substrate - Slowest Kinetic Rate has a relatively slower rate of color development and is a less sensitive substrate than ab171524 (TMB ELISA Substrate - Fast Kinetic Rate.

In general, one would select ab171527 (TMB ELISA Substrate - Slowest Kinetic Rate) if you feel the target protein detected in your assay is present in high amounts. The slow color development will allow you to stop the assay in a reasonable amount of time (eg. 5-10 minutes).

If you used ab171524 (TMB ELISA Substrate - Fast Kinetic Rate) as the substrate in the above case the color development may occur to quickly. This would lead to problems such as ODs quickly growing beyond the linear range of your spectrophotometer. In addition, while you are adding STOP solution to the first row of the plate, the remaining wells will continue to develop color while you add STOP solution to all the wells of the plate, leading to inconsistant results across the plate.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Aug 21 2013

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