Overview

  • Product name
    Anti-Diphtheria Toxin antibody
    See all Diphtheria Toxin primary antibodies
  • Description
    Rabbit polyclonal to Diphtheria Toxin
  • Host species
    Rabbit
  • Specificity
    ab53828 reacts both with toxoid and toxin.
  • Tested applications
    Suitable for: WB, ELISAmore details
  • Immunogen

    Diphtheria toxoid (formaldehyde inactivated Diphtheria toxin).

  • General notes

    For a recombinant antibody to Diphtheria Toxin, please see - ab209329.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Constituent: Whole serum
  • Purity
    Whole antiserum
  • Purification notes
    Sterile filtered rabbit serum.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab53828 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 dilution. Detects a band of approximately 63 kDa (predicted molecular weight: 58 kDa).
ELISA Use at an assay dependent dilution. Reacts well in ELISA with coated diphtheria toxoid.

Target

  • Relevance
    Corynebacterium diphtheriae is a gram-positive, nonmotile bacteria found in soil and animal feces. C. diphteriae infect the epithelial cells of the upper respiratory tract from where they produce and secrete a potent toxin. This toxin is absorbed and disseminated through lymph channels and blood to the susceptible tissues of the body.
  • Cellular localization
    Secreted
  • Alternative names
    • Corynebacterium diphtheriae toxoid antibody
    • Diphtheria Toxin antibody
    • tox antibody

References

This product has been referenced in:
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you very much for your reply.

I am sorry that these suggestions are not more helpful. If you would like to try a different DT antibody please let me know and I can arrange a testing discount for you.

If there is anything else that we can do for you, please don't hesitate to ask and I'll be happy to assist you.

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Question
Answer

Thank you very much for your reply and for sending this additional protocol information and the images.

I've looked through the images and alsodiscussed the situation with some of my colleagues. Based on these images, it looks like the antibody volume was held constant with increasing amounts of protein. All of the mouse antibody ab51882 seems to be binding to the beads, but the protein to antibody ratio needs to be optimized. We would recommend using a minimal amount of protein (1 ug or less) and holding this quantity constant while increasing the volume of the antibody, in order to find the optimal protein:antibody ratio. By using a higher quantity of purified protein, the antibody may be saturated so that it can not bind any more protein.

With the rabbit antibody ab53828 (I mis-typed ab53848 in my previous email), it looks like there may be too much protein loaded onto the gel. I'd suggest a similar protocol with a small amount of protein held constant with increasing amount of antibody. It's possible that the rabbit antibody has a higher binding affinity compared to the mouse antibody, and less of this antibody can be used.

As I mentioned before, we haven't tested any of our DT antibodies in IP so we aren't sure which will be most suitable for this application. If you would like to try one of our other DT antibodies, we do have a testing discount program that you can participate in. I might recommend the goat polyclonal ab19950 instead of one of the other monoclonals, as the polyclonal antibodies will have more binding sites on the protein.

I hope that this will be useful, but please keep me updated of any progress and let me know if you have any questions or if there is anything else that we can do for you, and I'll be happy to help.

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Question

I have a question regarding your Immunoprecipitation protocol and looking for some advice. We are looking to bind CRM197 which also contains Vi antigen, basically we are hoping to bind all of the CRM197 so we can detect the Vi.

What we have done is added 20ug of CRM197 with 1-50uL of MAB AB51882 (MAB to DT) overnight rocking at 4 degrees. The next day 100uL of Protein G Beads were added for 4 hours rocking at 4 degrees.

Then centrifuging at 14,000 rpm for 2 minutes, taking off the supernatant and resuspending the pellet (beads) in the same volume as the supernatant then adding 6x loading buffer (50uL) to both the supernatant and the resuspended pellet, heating for 5 minutes then running on a Coomassie. We are hoping by SDS page Coomassie Stain that all the CRM197 has bound to the beads and is not detected in the supernatant.Also we are only focusing on the binding of CRM197 at this time so there is only CRM197 in the sample

Since we are using CRM197 ,we are using PBS buffer only, although we have tried resuspending the beads in 400uL Lysis buffer.

We are following your protocol with a few exceptions:

Protein G Beads=

100mg Beads to 1mL PBS for 1 hour rocking at RT

Centrifuged at 14,000rpm for 2 minutes

Washed with 1mL PBS, centrifuged

Added 400uL PBS or Lysis buffer (non denaturing Lysis buffer- 20mM Tris-HCL pH 8.0, 137mM NaCl, 10% Glycerol, 2mM EDTA and 1% Triton X-100)

Procedure=

CRM197 + 2-25uL MAB AB51882 ( Also have tried Rabbit to DT AB53828, 2-50uL and mixing of both AB51882 and AB53828, 25uL of each) Overnight at 4 degrees rocking

Added 100uL of Beads x 4 hours at 4 degrees rocking

Centrifuged at 14,000 for 2 minutes

Removed supernatant and resuspended pellet in same volume as supernatant

Added 50uL of 6x Loading Buffer to both supernatant and resuspended pellet, heated at 100 degrees x 5 minutes

Ran on Coomassie

We have also tried binding the antibody to the beads overnight then adding 20ug of CRM197 at 4 degrees for 4 hours then following the procedure above.

Results- Band appears for both the supernatant and the pellet. Depending on the experiment the supernatant band is sometimes greater than the pellet. We are hoping to not to have a band on the Coomassie for the supernatant and a band that matches our control for the resuspended pellet.

Is there another antibody to use that may be better suited for this experiment or if you could give us any advise that may help us bind the CRM197 to the beads, it would be greatly appreciated

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Answer

Thank you very much for contacting us with your questions and for sending the details of your protocol.

I do have a few additional questions just to make sure that I understand the situation:

1) How is the CRM197 prepared prior to the IP? Is it in a denatured conformation?
2) In what buffer is the CRM197 incubated with the antibody? What is the total volume during this incubation (protein, antibody, and buffer)?
3) Is there a reason why the elution was done at 100C for 5 min instead of 50C for 10 min? Have you also tried a double elution?
4) Do you have any images of your results?
5) Have you tried using protein A antibodies with the rabbit polyclonal ab53848 (slightly higher affinity than protein G)?
6) Have the antibodies been tested against the CRM197 protein in a Western blot or ELISA?

We have only tested these antibodies in Western blot and ELISA, so we don't have a specifically optimized IP protocol for them, but I may have some suggestions after looking over the additional details. It is also possible that these antibodies are not suitable for use in IP. Unfortunatley none of our DT antibodies have been tested in IP so we don't know which would be best to use in your assay, however we do have a testing discount program available if you would like to try a differentDT antibody in IP.

I look forward to hearing from you. Please let me know if you have any further questions and I'll be happy to help.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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