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  1. Link

    alkaline-phosphatase-conjugation-kit-lightning-link-ab102850.pdf

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Cell Biology Cell Cycle Kinases/Phosphatases Phosphatases
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Alkaline phosphatase Conjugation Kit - Lightning-Link® (ab102850)

  • Datasheet
  • SDS
Reviews (1)Q&A (4)References (25)

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Western blot - Alkaline phosphatase Conjugation Kit;- Lightning-Link
  • Alkaline Phospahatse Conjugation Kit
  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling anti-bovine Hp antibody for ELISA
  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling anti P. falciparum circumsporozite protein antibody for ECL slot blot assay
  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling antibodies for sandwich ELISA
  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling MPC and MYSV6 polyclonal antibodies for microfluidic sandwich ELISA

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Overview

  • Product name

    Alkaline phosphatase Conjugation Kit - Lightning-Link®
    See all Alkaline phosphatase kits
  • Product overview

    An alternative Alkaline Phosphatase Conjugation Kit (Animal Free) - Lightning-Link® (ab269901) is now in stock. The alkaline phosphatase enzyme used in this kit are produced recombinantly (in vitro) for high batch-to-batch consistency and long term security of supply and scalability. The conjugation protocol, buffer requirements, storage conditions, and the product sizes are identical.


    Alkaline Phosphatase Conjugation Kit / Alkaline Phosphatase Labeling Kit ab102850 uses a simple and quick process for alkaline phosphatase labeling / conjugation of antibodies. It can also be used to conjugate other proteins or peptides. Learn about our antibody labeling kits and their advantages.


    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 alkaline phosphatase 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.


    Custom size conjugation kits up to 100 mg are available on demand. Please contact us to discuss your requirements.

  • Notes

    This product is manufactured by Expedeon, an Abcam company, and was previously called Lightning-Link® Alkaline Phosphatase Labeling Kit. 702-0005 is the same as the 100 µg size. 702-0010 is the same as the 3 x 100 ug size. 702-0030 is the same as the 3 x 10 ug size. 702-0015 is the same as the 1 mg size.

    Amount and volume of antibody for conjugation to Alkaline Phosphatase

     Kit size Recommended maximum 
    amount of antibody
    Maximum antibody 
    volume1
    3 x10 µg 3 x 10 µg 3 x 10 µL
    100 µg 1 x 100 µg  1 x 100 µL
    3 x 100 µg 3 x 100 µg  3 x 100 µL
    1 mg 1 x 1 mg 1 x 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 > 1 mg/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% BSA 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%

    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.

    Storing and handling conjugation kits

    Lyophilized Lightning-Link® components are hygroscopic.

    Kits are intentionally shipped at ambient temperature with silica gel to avoid exposure to moisture. Upon receipt, store the kit frozen and protect from moisture. Before opening the outer container, allow the lyophilized components to reach room temperature to minimize condensation.

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 mg 100 µg 3 x 10 µg 3 x 100 µg
    ab274118 - AP-Mix 1 x 1mg 1 x 100µg 3 x 10µg 3 x 100µg
    ab274119 - Modifier reagent 1 x 200µl 1 x 200µl 1 x 200µl 1 x 200µl
    ab274296 - Quencher reagent 1 x 200µl 1 x 200µl 1 x 200µl 1 x 200µl
  • Research areas

    • Cell Biology
    • Cell Cycle
    • Kinases/Phosphatases
    • Phosphatases
    • Tags & Cell Markers
    • Epitope Tags
    • Conjugates
    • Kits/ Lysates/ Other
    • Kits
    • Conjugation Kits
    • Alkaline phosphatase
  • Alternative names

    • AP

Associated products

  • Related Products

    • Antibody Concentration And Clean-Up Kit (ab102778)
    • Antibody Purification Kit (Protein A) (ab102784)

Images

  • Western blot - Alkaline phosphatase Conjugation Kit;- Lightning-Link
    Western blot - Alkaline phosphatase Conjugation Kit;- Lightning-LinkImage from Feng, Linyuan, et al., PLoS pathog., 14(1): e1006867; doi: 10.1371/journal.ppat.1006867. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Feng, Linyuan, et al used Alkaline phosphatase Conjugation Kit - Lightning-Link® (ab102850) as part of examining the interaction between UL23 and Nmi identified by coimmunoprecipitation. They used the kit to conjugate Alkaline phosphatase to primary antibodies for use in western blot.
    (A-B) Human U251 cells were co-transfected with a combination of two plasmids expressing FLAG- and HA-tagged proteins, and then harvested at 48 hours posttransfection. (C-D) Human U251 cells were infected with human cytomegalovirus (HCMV), a human herpesvirus, (MOI = 1) and cellular lysates were prepared at 48-72 hours postinfection. The input protein samples (80 µg) (Input) (lanes 3, 6, 9, 12, 15, 18, 21, and 24) and samples (15 µg) that were precipitated with anti-HA (IP (anti-HA)) (lanes 2, 5, 8, and 11), anti-FLAG (IP (anti-FLAG)) (lanes 1, 4, 7, and 10), anti-Nmi (lanes 13, 16, 19, and 22), anti-UL44 (lanes 14 and 17), or anti-UL23 antibodies (lanes 20 and 23), were separated on SDS-containing polyacrylamide gels, and assayed with Western blot analysis using anti-HA (anti-HA) (A), anti-FLAG (anti-FLAG) (B), anti-UL44 (anti-UL44) (C), anti-UL23 (anti-UL23) (D), and anti-Nmi (anti-Nmi) (C-D) antibodies that were directly conjugated to alkaline phosphatase using the Lightning-Link® kit ab102850, respectively.

  • Alkaline Phospahatse Conjugation Kit
    Alkaline Phospahatse Conjugation Kit

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

  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling anti-bovine Hp antibody for ELISA
    Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling anti-bovine Hp antibody for ELISAImage from Thomas FC et al., BMC Vet Res., 11, 207. Fig 1.; doi: 10.1186/s12917-015-0533-3. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Thomas FC et al used ab102850 as part of examining the toxicity of p17 protein assemblies.

    They used the kit to conjugate Alkaline phosphatase to anti-bovine Hp antibody for use in ELISA.

    Boxplot showing Hp concentration (μg/ml) in two SCC categories of composite milk samples * indicates an extreme value (values greater than 3 interquartile range (IQR) away from 25th or 75th percentile); IQR = 3rd quartile -1st quartile (represented by the height of the box). ° indicates an outlier value (values greater than 1.5 interquartile range (IQR) away from 25th or 75th percentile); IQR = 3rd quartile -1st quartile (represented by the height of the box).

  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling anti P. falciparum circumsporozite protein antibody for ECL slot blot assay
    Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling anti P. falciparum circumsporozite protein antibody for ECL slot blot assayImage from GrabiasB et al., PLoS One, 12(4): e0174229. Fig 3; doi: 10.1371/journal.pone.0174229. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Grabias B et al. used ab102850 as part of developing a rapid detection of Plasmodium falciparum infection in mosquitoes.

    They used the kit to conjugate Alkaline phosphatase to anti-Plasmodium falciparum circumsporozite protein antibody for use in ECL slot blot assay.


    Evaluation of whether conjugation of primary mAb 2A10 with alkaline phosphatase (1°-AP) enhanced sensitivity for the detection of Pfoocyst prepared from mosquito lysates when compared to the use of AP-conjugated secondary antibody (2°-AP). Detection limits were compared for each protocol by fitting the band intensities of serially diluted oocysts to a Michaelis-Menten regression curve and establishing a cutoff intensity threshold of mean + 2 SD from unfed mosquito specimens run on the same blot. Labeled primary antibody displayed overall higher band intensities across the range of oocyst dilutions examined and achieved lower limits of detection than the typical sandwich antibody format (0.009 oocyst versus 0.02 oocyst, respectively). The removal of an additional antibody incubation step also contributed to an overall shorter assay time in the newly developed slot blot protocol.

  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling antibodies for sandwich ELISA
    Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling antibodies for sandwich ELISAImage from Charlermroj R et al., PLoS One, 8(4): e62344. Fig 5; doi: 10.1371/journal.pone.0062344. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Charlermroj R et al. used ab102850 to run a sandwich ELISA and compare its sensitivity with a microsphere immunoassay based on Luminex using the same set of antibodies.

  • Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling MPC and MYSV6 polyclonal antibodies for microfluidic sandwich ELISA
    Alkaline phosphatase Conjugation Kit - Lightning-Link® labeling MPC and MYSV6 polyclonal antibodies for microfluidic sandwich ELISAImage from Thaitrong N et al., PLoS One, 8(12): e83231. Fig .; doi: 10.1371/journal.pone.0083231. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Thaitrong N et al. used ab102850 to run a microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. Nine different conditions for each disease panel were tested on the microfluidic platform using combinations of three concentrations of capture Ab (11E5, 2D6, and 5E7) and three concentrations of detection Ab (MPC-AP, MYSV6-AP).

Protocols

  • Protocol Booklet - Chinese Version
  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (25)

Publishing research using ab102850? Please let us know so that we can cite the reference in this datasheet.

ab102850 has been referenced in 25 publications.

  • Roth-Walter F  et al. Cow's milk protein ß-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells. J Allergy Clin Immunol 147:321-334.e4 (2021). PubMed: 32485264
  • Binder U & Skerra A PASylated Thymosin a1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology. Int J Mol Sci 22:N/A (2020). PubMed: 33374407
  • Davies G  et al. Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology. J Fungi (Basel) 7:N/A (2020). PubMed: 33396482
  • Salom D  et al. Human red and green cone opsins are O-glycosylated at an N-terminal Ser/Thr-rich domain conserved in vertebrates. J Biol Chem 294:8123-8133 (2019). PubMed: 30948514
  • Forsyth J  et al. Co-reactivity between related and unrelated environmental allergens in equine allergen-specific IgE serology testing in the UK. Vet Dermatol 30:544-e165 (2019). PubMed: 31464011
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
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1-5 of 5 Abreviews or Q&A

EasyLink AP Kit (ab102848)

Good Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted May 02 2014

Question

If I make 1 mg/mL with 100 ug of antibody in 100 uL volume as instructed, what would be the final concentration of the AP labeled antibody?

Read More

Abcam community

Verified customer

Asked on Jun 10 2013

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

Abcam Scientific Support

Answered on Jun 10 2013

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

Abcam community

Verified customer

Asked on May 31 2012

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

Abcam Scientific Support

Answered on Jun 01 2012

Question

Customer is trying to use the kit with Rb IgG, but is not getting a signal from the conjugated ab. Kit works well with ms IgM

Read More

Abcam community

Verified customer

Asked on May 31 2012

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

Abcam Scientific Support

Answered on May 31 2012

Question

Will a buffer concentration of 4mM phosphate be compatible with this conjugation kit?

Read More

Abcam community

Verified customer

Asked on Nov 04 2011

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.

Read More

Abcam Scientific Support

Answered on Nov 04 2011

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