Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553)
![Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Ki67 antibody [EPR3610]](https://www.abcam.com/ps/products/236/ab236553/Images/ab236553-481953-AlexaFluor488ConjugationKitFastLightningLinkimmunocytochemistryhap1humanki67.jpg "Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Ki67 antibody [EPR3610]")
Overview
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Product name
Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® -
Product overview
Alexa Fluor® 488 Conjugation Kit / Alexa Fluor® 488 Labeling Kit ab236553 uses a simple and quick process for Alexa Fluor 488 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 Alexa Fluor® 488 using this kit:
- add modifier to antibody and incubate for 15 mins
- add quencher and incubate for 5 mins
The Alexa Fluor® 488 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 Alexa Fluor® 488.
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® Rapid Alexa Fluor® 488 Labeling Kit. 332-0015 is the same as the 1 mg size. 332-0010 is the same as the 3 x 100 ug size. 332-0030 is the same as the 3 x 10 ug size. 332-0005 is the same as the 100 µg size.
Amount and volume of antibody for conjugation to Alexa Fluor® 488
Kit size Recommended
amount of antibody1Maximum
amount of antibodyMaximum antibody
volume23 x 10 µg 3 x 10 µg 3 x 20 µg 3 x 10 µL 100 µg 100 µg 200 µg 100 µL 3 x 100 µg 3 x 100 µg 3 x 200 µg 3 x 100 µL 1 mg 1 x 1 mg 1 x 2 mg 1 x 1 mL 1 Using the maximum amount of antibody may result in less labelling per antibody.
2 Ideal antibody concentration is 1mg/ml. 0.5 - 1 mg/ml can be used if the maximum antibody volume is not exceeded. Antibodies > 2mg/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% 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 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.
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.
Alexa Fluor® is a registered trademark of Molecular Probes, Inc, a Thermo Fisher Scientific Company. The Alexa Fluor® dye included in this product is provided under an intellectual property license from Life Technologies Corporation. As this product contains the Alexa Fluor® dye, the purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). As this product contains the Alexa Fluor® dye the sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: in manufacturing; (ii) to provide a service, information, or data in return for payment (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are sold for use in research. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5781 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@thermofisher.com.
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 µg 1 mg 3 x 10 µg 3 x 100 µg ab274037 - Alexa Fluor 488 1 x 100µg 1 x 1mg 3 x 10µg 3 x 100µg ab273994 - Modifier reagent 1 x 200µl 1 x 200µl 1 x 200µl 1 x 200µl ab273995 - Quencher reagent 1 x 200µl 1 x 200µl 1 x 200µl 1 x 200µl
Images
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Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Ki67 antibody [EPR3610]
Direct immunofluorescent staining using Recombinant Anti-Ki67 antibody [EPR3610] - BSA and Azide free (ab209897) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553).
Lightning-Link® Alexa Fluor® 488Anti-Ki67 antibody [EPR3610] conjugate was used to stain Ki67-wild-type HAP1 cells (top panel) and Ki67-knock-out HAP1cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 µg/ml of LL-Anti-Ki67 Alexa Fluor® 488 (shown in green) or ab209897 (Anti-Ki67 antibody, unlabelled control) overnight at +4°C. Ab209897 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red).
Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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![Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776]](https://www.abcam.com/ps/products/236/ab236553/Images/ab236553-481951-AlexaFluor488ConjugationKitFastLightningLinkimmunocytochemistryhap1human.jpg)
Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776]Direct immunofluorescent staining using Recombinant Anti-Vimentin antibody [EPR3776] - BSA and Azide free (ab193555) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553).
Lightning-Link® Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776] conjugate was used to stain Vimentin-wild-type HAP1 cells (top panel) and Vimentin-knock-out HAP1cells (bottom panel). The cells were fixed with 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 µg/ml of LL-Anti-Vimentin Alexa Fluor® 488 (shown in green) or ab193555 (Anti-Vimentin antibody, unlabelled control) overnight at +4°C. Ab193555 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red).
Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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![Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-EGFR antibody [E235]](https://www.abcam.com/ps/products/236/ab236553/Images/ab236553-481950-AlexaFluor488ConjugationKitFastLightningLinkimmunocytochemistrya431human.jpg)
Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-EGFR antibody [E235]Direct immunofluorescent staining using Recombinant Anti-EGFR antibody [E235] - BSA and Azide free (ab227459) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553).
Lightning-Link® Alexa Fluor® 488 Anti-EGFR antibody [E235] conjugate was used to stain wild-type A431 cells (top panel) and MCF7 negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h.
The cells were incubated with 1 µg/ml of LL-Anti-EGFR Alexa Fluor® 488 conjugate (shown in green) or ab227459 (Anti-EGFR antibody, unlabelled control) overnight at +4°C. Ab227459 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red).
Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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![Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Cytokeratin 19 antibody [EP1580Y]](https://www.abcam.com/ps/products/236/ab236553/Images/ab236553-481948-AlexaFluor488ConjugationKitFastLightningLinkimmunocytochemistrymcf7human.jpg)
Direct immunofluorescence - Lightning-Link® Alexa Fluor® 488 Anti-Cytokeratin 19 antibody [EP1580Y]Direct immunofluorescent staining using Recombinant Anti-Cytokeratin 19 antibody [EP1580Y] - BSA and Azide free (ab195872) labelled with Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553).
Lightning-Link® Alexa Fluor® 488 Anti-Cytokeratin 19 [EP1580Y] conjugate was used to stain wild-type MCF7 cells (top panel) and A431 negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) or 100% methanol at -20°C (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were incubated with 1 µg/ml of LL-Anti-Cytokeratin 19 Alexa Fluor® 488 (shown in green) or ab195872 (Anti-Cytokeratin 19 antibody, unlabelled control) overnight at +4°C. Ab195872 treated cells only were incubated with ab150087 at 1/1000 dilution for 1 hour at room temperature (shown in red).
Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Alexa Fluor® 488 Conjugation Kit (Fast)Image from Bardhan, Kankana, et al., Scientific reports, 9(1):17252. doi: 10.1038/s41598-019-53463-0. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/Bardhan, Kankana, et al used Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553) as part of examining PD-1pY248+ (pPD-1) expression in human T cells. They used the kit to conjugate Alexa Fluor® 488 to Anti-PD-1pY248 antibody for use in flow cytometry.
pPD-1 is predominantly expressed in CD8+ TCM cells. CCR7 and CD45RO markers were used to identify central memory (TCM) and effector memory (TEM) T cells. After gating on CD45RO expression, TCM (CD45RO+CCR7+) and TEM (CD45RO+CCR7−) CD4+ and CD8+ T cells were identified by assessing expression of CCR7. In TCM and TEM populations, expression of PD-1 was determined and, subsequently, expression of pPD-1 (pPD-1-Y248) was assessed in the PD-1+ population within each subset. Results are representative of six separate experiments. -
Alexa Fluor 488 Conjugation KitImage from Soldevila, Ferran et al. Frontiers in immunology vol. 9 1800. 15 Aug. 2018, doi:10.3389/fimmu.2018.01800. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/Soldevila, Ferran et al used Alexa Fluor® 488 Conjugation Kit - Lightning-Link® (ab236553) as part of examining myeloid cells in the porcine palatine tonsil. They used the kit to conjugate Alexa Fluor® 488 to anti-pig CD4α antibody for use in confocal microscopy.
In situ localization of the CD14+ cells and plasmocytoid dendritic cells in porcine palatine tonsil. CD14+ cells and pDCs were localized by confocal microscopy following ethanol fixation of tonsil slices. The areas assessed included the follicle (F), the interfollicular region (IFA), the crypt (C). The tissue was stained using panel 1 antibodies; white arrow CD14+ cells and yellow arrow pDCs. Images are representative of at least two images from each section, from three different pigs. Objective used: (A) 63× oil immersion. Scale bars as shown. -
Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link®A polyclonal antibody was purchased from a commercial source and conjugated to Alexa Fluor® 488 using Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553) and to CF™488A dye using a competitor conjugation kit. The conjugates were tested by ELISA and the fluorescence measured at 523nm after excitation at 487nm. The Abcam conjugate out-performs the competitor.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (12)
ab236553 has been referenced in 12 publications.
- Ma J et al. SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage. EMBO J 40:e108249 (2021). PubMed: 34296442
- Miralles de Imperial-Ollero JA et al. An in vivo model of focal light emitting diode-induced cone photoreceptor phototoxicity in adult pigmented mice: Protection with bFGF. Exp Eye Res 211:108746 (2021). PubMed: 34450185
- Wu XY et al. Complement C1q synergizes with PTX3 in promoting NLRP3 inflammasome over-activation and pyroptosis in rheumatoid arthritis. J Autoimmun 106:102336 (2020). PubMed: 31601476
- Kraman M et al. FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1, Enhances T-Cell Activation Resulting in Potent Antitumor Activity. Clin Cancer Res N/A:N/A (2020). PubMed: 32299814
- Kräutler NJ et al. Quantitative and Qualitative Analysis of Humoral Immunity Reveals Continued and Personalized Evolution in Chronic Viral Infection. Cell Rep 30:997-1012.e6 (2020). PubMed: 31995768