Goat Anti-Mouse IgA alpha chain (DyLight® 650) (ab97014)

Overview

  • Product name
    Goat Anti-Mouse IgA alpha chain (DyLight® 650)
    See all IgA secondary antibodies
  • Host species
    Goat
  • Target species
    Mouse
  • Specificity
    By immunoelectrophoresis and ELISA this antibody reacts specifically with Mouse IgA. Cross reactivity with other immunoglobulins and light chains is less than 0.1%.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, Flow Cytmore details
  • Conjugation
    DyLight® 650. Ex: 654nm, Em: 673nm

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C.
  • Storage buffer
    Preservative: 0.09% Sodium azide
    Constituents: 0.2% BSA, PBS
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Antiserum was solid phase adsorbed to ensure class specificity. This antibody was isolated by affinity chromatography using antigen coupled to agarose beads and conjugated to DyLight® 650.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • General notes
    DyLight® 650 replaces DyLight® 649.
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab97014 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
IHC-P 1/50 - 1/500.

DyLight® 650 replaces DyLight® 649. The DyLight® 650 excitation is 652nm and the emission is 672nm. DyLight® 650 hence provides the same far-red fluorescence and photostability as the DyLight® 649 dye. Please refer to the vial in order to check whether the product you received is DyLight® 649 or DyLight® 650. This information however will have no impact on your experiments. For more information please refer to DyLight® 650.

ICC/IF 1/50 - 1/500.
Flow Cyt 1/50 - 1/200.

Images

  • Emission spectra of DyLight® fluorochromes available in our catalog.
    Line colors represent the approximate visible colors of the wavelength maxima.

References

ab97014 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Thank you for your updates and for your co-operation in this matter.

I am glad that the source of the problem has been identified.

If you need any further assistance in the future, please do not hesitate to contact me.

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Question

oM of our costumers is having problems with:

ABCAM Goat polyclonal anti-mouse IgA alpha chain - DyLight 650

Code ab97014

Batch: GR76514-1

P.O. # 0076/12 (March 2012)



She is using ICC/ IF technique and says that the antibody isn't giving the desired signal. It's negative or too weak on her cell preparations (see attached images).





Protocol tested with ab97014:



1- For cell culture were used 24 well plates with one 2% gelatin covered circular glass coverslip inside each well.



2- Cells from CACO-2 line (human epithelial colorectal adenocarcinoma cells) were cultivated for 16 Hours in 1 mL of DMEM with 10% FBS Cultire Medium per well.

At that stage, approximated 10000 cells were in the culture. This number of cells is not confluent, so allow to take pictures of isolated cell groups (2 to 5 cells only).



3- After 16 Hours, cells were washed with PBS (1X) and fixed with 3% Formalin for 10 minutes.



4- Washes with PBS (3X).



5- Cell permeabilization: 0.05% Saponin/PBS for 10 minutes.



6- Washes with PBS (3X).



7- Protein block: 1% FBS in PBS for 10 minutes.



8- Primary antibody: Monoclonal mouse anti-ADRP (Fitzgerald) diluted 1:75 in 0.05% Saponin/PBS for 1 hour, at room temperature.



9- Washes with PBS (3X).



10- Secundary antibody: ab97014 diluted 1:50 in 0.05% Saponin/PBS for 45 minutes, at room temperature.



11- Washes with PBS (3X).



12- Bodipy (488 nm) incubation for 45 minutes, at room temperature.



13- Washes with PBS (3X).



14- DAPI incubation for 5 minutes, at room temperature.



15- Wash and mounting of the coverslips on glass slides.



16- Slides were analyzed on Confocal Microscope (Fluoview FV10i Olympus)





NOTES:



1- As a positive reaction control the same conditions exposed above were also used with secundary antibody anti-mouse Rhodamine from KPL manufacturer and the reaction signal was OK.



2- Some variations of the protocol mentioned above have already been tested with ab97014 all without success:

- Increase permeabilization time using 0.05% Saponin (Protocol - Step 5): 30 minutes

- Change permeabilization reagent (Protocol - Step 10): 0.2% Triton/ PBS

- Change permeabilization reagent (Protocol - Steps 5 and 10): 0.2% Triton/ PBS

Even with these protocol changes, the reaction signal was still negative or too weak.





What do you think about the case?


1-) Could this be a problem with the reagent vial (ab97014 - Batch GR76514-1)?

2-) If you think the antibody is OK, can you give us any clue to improve protocol?

I'm forward to hearing from you.

Thanks for your attention.

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Answer

rrding ab97014 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this secondary antibody.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

1) Primary antibody - Could you specify the followings:

- Isotype - Is it compatible with the secondary antibody?

- Purification?

- Has it been tested for ICC/IF on human cells?

2) Permeabilization:

Has the customer applied any other permeabilzing agents such as acetone or TBS-T?

3) Positive control:

Has any positive control be applied parallel with the samples?

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

I look forward to hearing from you soon.

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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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