Overview

  • Product name
    Alkaline phosphatase Conjugation Kit
  • Product overview

    Alkaline Phosphatase Conjugation Kit ab102850 uses a simple and quick process to conjugate an antibody to Alkaline Phosphatase. It can also be used to conjugate other proteins or peptides.


    To conjugate an antibody to Alkaline Phosphatase using this kit:
    - add modifier to antibody and incubate for 3 hrs
    - add quencher and incubate for 30 mins
    The conjugated antibody can be used immediately in WB, ELISA, IHC etc. No further purification is required and 100% of the antibody is recovered for use.


    Learn about buffer compatibility below; for incompatible buffers and low antibody concentrations, use our rapid antibody purification and concentration kits. Use the FAQ to learn more about the technology, or about conjugating other proteins and peptides to HRP.


     


     

  • Notes

    Amount and volume of antibody for conjugation to Alkaline Phosphatase

     Kit size   Recommended maximum 
    amount of antibody
    Maximum antibody 
    volume1
    30 µg   3 x 10 µg 3 x 10 µL
    100 µg   100 µg  100 µL
    300 µg   3 x 100 µg  3 x 100 µL
    1 mg   1 mg 1 mL

    1 Ideal antibody concentration is 1mg/ml. 0.5 - 1 mg/ml can be used if the maximum antibody volume is not exceeded. Antibodies > 5mg/ml or < 0.5 mg/ml should be diluted /concentrated.

     

    Buffer Requirements for Conjugation

    Buffer should be pH 6.5-8.5.

    Compatible buffer constituents 
    If a concentration is shown, then the constituent should be no more than the concentration shown. If several constituents are close to the limit of acceptable concentration, then this can inhibit conjugation.

    50mM / 0.6% Tris1 0.1%/1% BSA2 50% glycerol
    0.1% sodium azide PBS Potassium phosphate
    Sodium chloride HEPES Sucrose
    Sodium citrate EDTA Trehalose

    1 Tris buffered saline is almost always ≤ 50 mM / 0.6%
    2 1% BSA gives lower quality conjugates, BSA can also interfere with the use of the conjugated antibody in tissue staining.

    Incompatible buffer constituents

    Thiomerosal Proclin Glycine
    Arginine Glutathione DTT

    If a constituent of the buffer containing your antibody or protein is not listed above, please check the FAQ or contact us.

    Only purified antibodies are suitable for use, ie. where other proteins, peptides, or amino acids are not present: antibodies in ascites fluid, serum or hybridoma culture media are incompatible.

  • Tested applications
    Suitable for: Conjugationmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab102850 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
Conjugation Use at an assay dependent dilution.

Images

  • This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.

Protocols

References

This product has been referenced in:
  • Feng L  et al. Human cytomegalovirus UL23 inhibits transcription of interferon-? stimulated genes and blocks antiviral interferon-? responses by interacting with human N-myc interactor protein. PLoS Pathog 14:e1006867 (2018). Read more (PubMed: 29377960) »
  • Lo Passo C  et al. Molecular characterization of two sub-family specific monoclonal antibodies to meningococcal Factor H binding protein. Heliyon 4:e00591 (2018). Read more (PubMed: 29644339) »
See all 3 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

EasyLink AP Kit (ab102848)

Good Excellent 5/5 (Ease of Use)
Abreviews
The kit is easy to use and achieve its purpose.
I have one suggestion: to add some kind of detection, after conjugation is finished.
This will able the customer to detect the success of AP conjugation, before using the AP conjugated protein in the intended protocol.

Tested by ELISA

Abcam user community

Verified customer

Submitted May 02 2014

Answer

The conjugation protocol requires the initial antibody volume be diluted 20% during the conjugation reaction. 1/10 antibody volume of EL-Modifier is added at the beginning of the reaction and antibody 1/10 volume of EL-Quencher is added at the conclusion of the conjugation.

Read More

Question

Please find your answers inline.

- What were the antibody volume, amount and concentration? – 100μl of a 1mg/ml antibody concentration used for conjugation

- What was the antibody buffer (including any additives/stabilisers)? – PBS pH7.4 (GIBCO/Invitrogen); no additive/stabilizer

- How had the antibody been purified? – Two steps (1) 45% Ammonium sulfate cut of hyperimmune Rabbit serum; (2) 35% Ammonium sulfate cut of material at step 1. Final precipitate dissolved in PBS, and dialyzed extensively against PBS. Antibody checked by both indirect and sandwich ELISAs. Concentration determined by ELISA using a serially diluted Rabbit IgG standard (Millipore) on a plate coated with goat anti-rabbit IgG (Southern Biotech) and detected with the corresponding AP-conjugate of the goat antibody.

- The antibody datasheet would be useful, if there is one. – I don’t understand what this means.

- What were the reagent volumes, times and temperatures used? – 100μl antibody pipette-mixed with 10μl EL-modifier; put over the lyophilized material, pipette-mixed gently; RT (23oC) incubation for 3h and 10 minutes; 10μl of EL-Quencher added, incubated for 30 minutes at RT.


This protocol, used the first time, gave us inefficient conjugation with Rabbit IgG with one lot of Abcam reagent; another lot, sent for us to try, gave absolutely no AP signal, indicating no conjugation. The antibody, however, did bind to the antigen, since a goat anti-Rabbit IgG-HRP (applied after washing off the AP substrate) followed by TMB substrate gave the expected color.

Read More
Answer

Thank you for your reply and for providing that information.

I believe the cause of the problem is the purity of the rabbit antibody. As the antibody is purified using ammonium sulphate and not Protein A/G, then the sample of antibody is not pure enough for the EasyLink kit to work efficiently.

What I could do, is to send one of our antibody purification kits (ab102783), which could help resolve this issue and then you could use the purified antibody with the conjugation kit. If you do not wish to use this method, then I would be happy to refund the cost of your original purchase, so that you could try the conjugation using an alternative method.

Please let me know how you would like to continue.

Read More

Answer

Thank you for contacting Abcam yesterday.

I have talked to the lab about the issue you have been having and our kits are designed primarily for labeling IgGs; the failed conjugation would not have been because rabbit IgG was used. They have asked for some more information:

- What were the antibody volume, amount and concentration?
- What was the antibody buffer (including any additives/stabilisers)?
- How had the antibody been purified?
- The antibody datasheet would be useful, if there is one.
- What were the reagent volumes, times and temperatures used?

If you could provide this information, then this would help us track down the cause of the problems you are having with the kit.

I look forward to your reply.

Read More

Question
Answer

Thank you for contacting Abcam. The 4mM phosphate buffer concentration will work for ab102850. Also the antibody must be purified and at a concentration of 1mg/ml and additives such as BSA and azide can reduce conjugation efficiency.   If there is anything else I can help you with, please let me know.

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