ECL Substrate Kit (Very High Sensitivity) (ab133408)

Submit a review Q&A (5)References (1)
Western Blot 1/50000 primary antibody, 1/5000 secondary antibody, 10 second exposure
  • Western Blot 1/25000 primary antibody, 1/5000 secondary antibody, 10 second exposure

Overview

  • Product name

    ECL Substrate Kit (Very High Sensitivity)
    See all ECL Substrate kits

  • Product overview

    Very High Sensitivity ECL Substrate Kit () ab133408 uses a new horseradish peroxidase (HRP) substrate specially developed to take advantage of the large linear dynamic range provided by CCD imaging. It is designed for the detection of proteins from 4.6pg to 4.7ng of protein per band.


    ECL Substrate Kit ab133408 produces a strong, extremely long-lived signal, which, combined with very low background levels, allows for long exposure times enabling the detection of low-abundance proteins. Additionally, the signal from the ECL substrate is linear with respect to protein amount over a broad range of concentrations, displaying no substrate depletion at high protein loads, allowing the user to take full advantage of the linear range of the CCD detection method.


    The ECL substrate is also compatible with X-ray film detection, though the limited dynamic range of film will make resulting data less quantitative.


    Our ECL kits include our popular ECL Substrate Kit ab65623 and our high sensitivity ECL substrate kits:
    - High Sensitivity ECL Substrate Kit ab133406 to detect 23pg-187ng of protein per band
    - this kit (Very High Sensitivity ECL Substrate Kit ab133408) to detect 4.6pg-4.7ng of protein per band
    - Ultra High Sensitivity ECL Substrate Kit ab133409 to detect 1.2pg-2ng of protein per band 


    Detection ranges in pg and ng stated above should be used for guidance only as detection range is dependent on the molecular weight of a protein.


    This product was previously called Optiblot ECL Max Detect Kit (4.6pg-4.7ng).

  • Notes

    Use:
    - 100ml kit for 1000cm2 membrane
    - 200ml kit for 2000cm2 membrane

    The primary antibody can often be diluted 5 to 10 fold more than usual when using this ECL substrate. A typical primary antibody dilution range using the substrate is 1/5000 - 1/20,000, with a typical secondary antibody dilution range of 1/20,000 - 1/100,000. Some optimisation may be required.

  • Tested applications

    Suitable for: WBmore details

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab133408 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
WB
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration.

Images

  • Western Blot 1/50000 primary antibody, 1/5000 secondary antibody, 10 second exposure
    Western Blot 1/50000 primary antibody, 1/5000 secondary antibody, 10 second exposure

    Each lane contains the following amount of COX2 recombinant protein (ab58868): 1) 100ng 2) 50ng 3) 25ng 4) 12.5ng 5) 6.25ng 6) 3.13ng 7) 1.56ng 8) 780pg 9) 390pg 10) 195pg 11) 98pg. The blot was probed with rabbit anti-COX2 antibody (ab15191) at 1/50000 dilution and with a rabbit secondary (ab97080) at 1/5000 dilution. The blot was developed with 20ml of Optiblot ECL Max Detect.

    Exposure time: 10 seconds

  • Western Blot 1/25000 primary antibody, 1/5000 secondary antibody, 10 second exposure
    Western Blot 1/25000 primary antibody, 1/5000 secondary antibody, 10 second exposure

    Each lane contains the following amount of COX2 recombinant protein (ab58868): 1) 100ng 2) 50ng 3) 25ng 4) 12.5ng 5) 6.25ng 6) 3.13ng 7) 1.56ng 8) 780pg 9) 390pg 10) 195pg 11) 98pg. The blot was probed with rabbit anti-COX2 antibody (ab15191) at 1/25000 dilution and with a rabbit secondary (ab97080) at 1/5000 dilution. The blot was developed with 20ml of Optiblot ECL Max Detect.

    Exposure time: 10 seconds

References (1)

Publishing research using ab133408? Please let us know so that we can cite the reference in this datasheet.

ab133408 has been referenced in 1 publication.

  • Wege S & Poirier Y Expression of the mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) in tobacco leaves leads to phosphate export. FEBS Lett 588:482-9 (2014). PubMed: 24374333

Customer reviews and Q&As

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1-5 of 5 Abreviews or Q&A

Question

Can any of Abcam's ECL HRP subtrates be used for ELISA?

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Answer

ab133409 has been used for ELISA applications.

However, since there are many different readers, plates, etc., testing/evaluation is necessary to assess utility for your specific experimental conditions.

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Question

is there a cutoff for developing? how long after i add the reagent will the signal be good for?

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Answer

Thank you for contacting us.

We recommend developing, i.e. incubation with the substrate for 2 to 5 minutes. Longer incubation does not improve the performance, but it does not harm either in most cases. If the amount of protein detected is too high (signal very strong) incubation should not be longer than 5 minutes and imaging should be done quickly after incubation to maintain the best liner dynamic range.

For average intensities, signal lasts for several hours. If careful precautions are taken to prevent the membrane from drying out, signal can last as long as 24 hours.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

concerning your new Optiblot ECL products line is there amont these kits a product comparable to SuperSignal West Dura Chemiluminescent Substrate (http://www.piercenet.com/browse.cfm?fldID=01041102) in terms of long lasting signal?
This info is very important for us for switching customer from Dura to your ECL.

Thanks in advance and kind regards

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Answer

Thank you for contacting us.

The product comparable to Super Signal west Dura is Optiblot ECL Max

https://www.abcam.com/optiblot-ecl-max-detect-kit-46pg-47ng-ab133408.html

Product Optiblot ECL Ultra ab133409 is even more sensitive than Max

https://www.abcam.com/optiblot-ecl-ultra-detect-kit-12pg-2ng-ab133409.html

Please recommend these to customers.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

Which of your products is comparable to Amersham ECL Prime Western Blotting Detection Reagent (RPN2232)

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Answer

Thank you for contacting us.

ab133408 is compatible, however it would be better than Amersham Prime.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

Inquiry: I would like to know if the ECl Ultra detect kit is compatible with fluorescent imagers, such as the Typhoon imagers. Furthermore, I am currently using the ECL2 (formerly ECL Plus) detection kit, and I would like a small sample in order to compare to what I am currently using. Is that possible? Thank you for your help.

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Answer

Thank you for your inquiry.

Since you need a suitable ECL for imagers, I suggest to use ab133408 (Optiblot ECL Max Detect Kit (4.6pg-4.7ng)) which also corresponds with the ECL plus you are using at the moment.

ab133408 has been validated for CCD/laser use and generates a linear and quantitative signal that is very stable.

https://www.abcam.com/ECL-Substrate-Kit-Very-High-Sensitivity-ab133408.html (or use the following: https://www.abcam.com/ECL-Substrate-Kit-Very-High-Sensitivity-ab133408.html).

Please note that stripping the membrane is not not recommended.

ab133409 (Optiblot ECL Ultra Detect Kit (1.2pg-2ng)) is also suitable but is much more sensitive. This means that with average expression levels of proteins the dilution factor of the antibodies has to be newly optimised (higher dilution factor).

Unfortunately, we do not have samples sizes of our product since this is logistically not feasible with over 95000 products.

But I would like to confirm that our products are covered by our Abpromise for six month after purchase.

https://www.abcam.com/abpromise

I hope this information is helpful and wish you good luck with your experiments.

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