Alkaline phosphatase Conjugation Kit - Lightning-Link® (ab102850)
Overview
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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.
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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 antibodyMaximum antibody
volume13 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
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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
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Alternative names
- AP
Associated products
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Related Products
Images
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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 KitThis illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.
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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).
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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 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.
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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
Datasheets and documents
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SDS download
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Datasheet download
References (25)
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