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ReadiLink™ xtra Rapid Cy5 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra rapid antibody labeling kits require essentially only 2 simple mixing steps without a column purification needed. Specially formulated Cy5 used in this ReadiLink™ kit is quite stable and shows good reactivity and selectivity with antibodies. The kit has all the essential components for labeling ~2x50 ug antibody. Each of the two vials of specially formulated Cy5 dye provided in the kit is optimized for labeling ~50 µg antibody. ReadiLink™ xtra Cy5 rapid antibody labeling kit provides a convenient and robust method to label monoclonal and polyclonal antibodies with the red fluorescent Cy5 fluorophore. Cy5 is one of the most used fluorophores for labeling antibodies. 
Figure 1. Overview of the ReadiLink™ xtra Rapid Antibody Labeling protocol. In just two simple steps, and with no purification necessary, covalently label microgram amounts of antibodies in under an hour.
Example protocol
AT A GLANCE
Important
Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following protocol is for recommendation.PREPARATION OF WORKING SOLUTION
Protein working solution (Solution A)
For labeling 50 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 5 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 50 µL of the target protein solution.Note If you have a different protein concentration, adjust the protein volume accordingly to make ~50 µg of protein available for your labeling reaction.
Note For labeling 100 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 10 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 100 µL of the target protein solution.
Note The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4; if the protein is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (cat# UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) with 0.1 to 0.5 % will be labeled well.
Note For optimal labeling efficiency, a final protein concentration range of 1 - 2 mg/mL is recommended, with a significantly reduced conjugation efficiency at less than 1 mg/mL.
SAMPLE EXPERIMENTAL PROTOCOL
Run conjugation reaction
- Add the protein working solution (Solution A) to ONE vial of labeling dye (Component A), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
Note If labeling 100 µg of protein, use both vials (Component A) of labeling dye by dividing the 100 µg of protein into 2 x 50 µg of protein and reacting each 50 µg of protein with one vial of labeling dye. Then combine both vials for the next step. - Keep the conjugation reaction mixture at room temperature for 30 - 60 minutes.
Note The conjugation reaction mixture can be rotated or shaken for longer time if desired.
Stop Conjugation reaction
- Add 5 µL (for 50 µg protein) or 10 µL (for 100 µg protein) which is 10% of the total reaction volume of TQ™-Dyed Quench Buffer (Component C) into the conjugation reaction mixture; mix well.
- Incubate at room temperature for 10 minutes. The labeled protein (antibody) is now ready to use.
Storage of Protein Conjugate
The protein conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). For longer storage, the protein conjugates could be lyophilized or divided into single-used aliquots and stored at ≤ –20 °C.Spectrum
Product family
References
View all 50 references: Citation Explorer
Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.
Authors: Nödling, Alexander R and Mills, Emily M and Li, Xuefei and Cardella, Davide and Sayers, Edward J and Wu, Shih-Hsiung and Jones, Arwyn T and Luk, Louis Y P and Tsai, Yu-Hsuan
Journal: Chemical communications (Cambridge, England) (2020): 4672-4675
Authors: Nödling, Alexander R and Mills, Emily M and Li, Xuefei and Cardella, Davide and Sayers, Edward J and Wu, Shih-Hsiung and Jones, Arwyn T and Luk, Louis Y P and Tsai, Yu-Hsuan
Journal: Chemical communications (Cambridge, England) (2020): 4672-4675
Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye-Protein Interaction.
Authors: Hensbergen, Albertus W and Buckle, Tessa and van Willigen, Danny M and Schottelius, Margret and Welling, Mick M and van der Wijk, Felicia A and Maurer, Tobias and van der Poel, Henk G and van der Pluijm, Gabri and van Weerden, Wytske M and Wester, Hans-Jürgen and van Leeuwen, Fijs W B
Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (2020): 234-241
Authors: Hensbergen, Albertus W and Buckle, Tessa and van Willigen, Danny M and Schottelius, Margret and Welling, Mick M and van der Wijk, Felicia A and Maurer, Tobias and van der Poel, Henk G and van der Pluijm, Gabri and van Weerden, Wytske M and Wester, Hans-Jürgen and van Leeuwen, Fijs W B
Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (2020): 234-241
Direct Cyanine-dCTP Labeling of DNA for Microarrays.
Authors: Rando, Oliver
Journal: Cold Spring Harbor protocols (2019)
Authors: Rando, Oliver
Journal: Cold Spring Harbor protocols (2019)
Direct Cyanine-dUTP Labeling of RNA for Microarrays.
Authors: Rando, Oliver
Journal: Cold Spring Harbor protocols (2019)
Authors: Rando, Oliver
Journal: Cold Spring Harbor protocols (2019)
Electrochemiluminescent aptasensor based on resonance energy transfer system between CdTe quantum dots and cyanine dyes for the sensitive detection of Ochratoxin A.
Authors: Gao, Jingwen and Chen, Ziyi and Mao, Lebao and Zhang, Wei and Wen, Wei and Zhang, Xiuhua and Wang, Shengfu
Journal: Talanta (2019): 178-183
Authors: Gao, Jingwen and Chen, Ziyi and Mao, Lebao and Zhang, Wei and Wen, Wei and Zhang, Xiuhua and Wang, Shengfu
Journal: Talanta (2019): 178-183
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