Cy5® Conjugation Kit (Fast) - Lightning-Link® (ab188288)

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conjugation
  • Cy5® Conjugation Kit - Lightning-Link® labeling NK2A peptide for confocal microscopy

Overview

  • Product name

    Cy5® Conjugation Kit (Fast) - Lightning-Link®

  • Product overview

    Cy5® Conjugation Kit / Cy5® Labeling Kit (ab188288) uses a simple and quick process for Cy5 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 Cy5 using this kit:
    - add modifier to antibody and incubate for 15 min
    - add quencher and incubate for 5 mins
    The Cy5 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 Cy5.


    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® Rapid Cy5 Labeling Kit. 342-0005 is the same as the 100 µg size. 342-0010 is the same as the 3 x 100 ug size. 342-0030 is the same as the 3 x 10 ug size. 342-0015 is the same as the 1 mg size.

    Amount and volume of antibody for conjugation to Cy5

     Kit size Recommended 
    amount of antibody    1     		 Maximum 
    amount of antibody    		 Maximum antibody 
    volume2
    3 x 10 µg 3 x 10 µg  3 x 20 µg 3 x 10 µL
    100 µg 1 x 100 µg 1 x 200 µg 1 x 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.

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 mg 100 µg 3 x 10 µg 3 x 100 µg
    ab274058 - Cyanine Dye5 1 x 1mg 1 x 100µg 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

  • Research areas

Images

  • conjugation
    conjugation
  • Cy5® Conjugation Kit - Lightning-Link® labeling NK2A peptide for confocal microscopy
    Cy5® Conjugation Kit - Lightning-Link® labeling NK2A peptide for confocal microscopyImage from Dassanayake RP et al., PloS One, 12(8):e0183610. Fig 4.; doi:10.1371/journal.pone.0183610. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Dassanayake, Rohana P et al used ab188288 as part of examining the antimicrobial activity of bovine NK-lysin-derived peptides.

    They used the kit to conjugate Cy5® to NK2A peptide for use in confocal microscopy.

    H. somni was incubated with unconjugated Cy5 (A) or 20 μM NK2A-Cy5 conjugate for 30 min and bacterial nuclear contents were stained with DAPI. NK2A-Cy5 conjugate is shown in red and DAPI is shown in blue.

References (4)

Publishing research using ab188288? Please let us know so that we can cite the reference in this datasheet.

ab188288 has been referenced in 4 publications.

  • Mellal K  et al. Immunometabolic modulation of retinal inflammation by CD36 ligand. Sci Rep 9:12903 (2019). PubMed: 31501473
  • Katoh H  et al. Immunogenetic Profiling for Gastric Cancers Identifies Sulfated Glycosaminoglycans as Major and Functional B Cell Antigens in Human Malignancies. Cell Rep 20:1073-1087 (2017). PubMed: 28768193
  • Rizvi AH  et al. Single-cell topological RNA-seq analysis reveals insights into cellular differentiation and development. Nat Biotechnol 35:551-560 (2017). PubMed: 28459448
  • Yuan S  et al. Changes in anti-thyroglobulin IgG glycosylation patterns in Hashimoto's thyroiditis patients. J Clin Endocrinol Metab 100:717-24 (2015). PubMed: 25380293

Customer reviews and Q&As

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Question

I have recently purchased (ab188288 – Cy5 Fast Conjugation Kit) to label an Antibody for a flowcytometry. But the storage buffer of the antibody contains 1XPBS, 1%BSA, 20% Glycerol (pH7). And 0.01% Thimerosal was added as a preservative, would this has an effect on the conjugation. Should I do dialysis for the antibody before labelling?

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Answer

Only the BSA seems to be a problem, its concentration should be 0.1%-0.5%. The ideal way is to remove BSA, we have kit for this https://www.abcam.com/BSA-Removal-Kit-ab173231.html. Alternatively you can give it a go however it might affect the efficiency of the kit. Please check the information on booklet;

10.2 Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g.EDTA), and sugars have no effect on conjugation efficiency. Azide (0.02 to 0.1%) and BSA (0.1 to 0.5%) have little or no effect. Glycerol up to 50% has no effect.

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Question

Are fluorescent conjugation kits (Cy5 labeling kit) suitable for the labeling of M13 bacteriophage through amine group?

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Answer

M13 bacteriophages have not been tested with our labeling kits. However, as long as it contains some primary amines which are available for labeling, then there should be no reason why this would not work. As for antibodies, it is important that the buffer does not contain any free amines or other unsuitable substances. Please see below for details of (un)compatible formulations:


Additives such as salts (e.g NaCl), sugars (e.g. sucrose) and chelators (e.g. EDTA) have no effect on the labeling reaction. We recommend avoiding nucleophiles such as amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol) that could interfere with the conjugation reaction.

Compatible additives:
up to 20mM Tris
up to 0.1% BSA (BSA up-to 1% will work but yield lower quality conjugates)
up to 0.1% gelatin
up to 0.1% sodium azide
PBS pH6.5-8.5
up to 50% glycerol
0.15M sodium chloride
50mM HEPES
0.02M potassium phosphate
0.001% Tween
Proclin 300
5% Trehalose


Incompatibile additives:
Tris above 20mM
Urea
50mM Imidazole
Glycine
Ethanolamine
DTT
Mercaptoethanol


Finally, the M13 bacteriophage will be a different molecular weight to that of an antibody, and the protocol will therefore have to be adjusted to allow for the size difference. It is difficult for us to provide any advice, as I do not know the MW of the bacteriophage, but I would generally recommend that you carry out a titration of different concentrations, in order to determine the best set-up for your assay.

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