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ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit *Production Scale*

ReadiLink™ Rapid Antibody Labeling Kits, designed for production scale, provide a convenient and efficient method for labeling large volumes of antibodies with our superior iFluor® dyes, XFD dyes (equivalent to Alexa Fluor®), and various other labels. These kits utilize reactive fluorophores modified with succinimidyl ester (SE) functional groups, which selectively bind to primary amines on proteins, resulting in remarkably bright and photostable conjugates. Every kit contains all the necessary components for three distinct labeling reactions and features a user-friendly, pre-packed spin column for efficient dye removal, maximizing conjugate yield. Each vial of iFluor® 488 SE dye provided in the kit is precisely formulated to label 1 mg of purified protein or antibody. Before labeling, it is important to remove stabilizing proteins like BSA from the sample and refrain from using amine-rich buffers like Tris, which might disrupt the labeling process. iFluor® 488 SE is a bright green-fluorescent dye with excitation and emission maxima of ~491 nm and ~516 nm, making it an excellent alternative to FITC and Alexa Fluor® 488 (Alexa Fluor® is the trademark of Invitrogen). With ReadiLink™ Rapid Antibody Labeling kits, researchers can directly label primary antibodies, eliminating the need for secondary antibodies and enhancing panel-building flexibility.

Example protocol

AT A GLANCE

Key Parameters for Optimal Results
  1. 1.0 mg Antibody (MW ~150 kDa)

  2. Antibody concentration: 2.0 mg/mL

  3. Antibody volume: 500 µL

SAMPLE EXPERIMENTAL PROTOCOL

Important

Before opening the vials, warm all components and briefly centrifuge. Immediately prepare necessary solutions before starting conjugation. This protocol is a recommendation.

Antibody Labeling Reaction
  1. Warm up a vial of reactive dye (Component A) to room temperature.

    Note: Each vial of reactive dye contains an optimized amount of dye to label 1 mg of IgG (MW ~150 kDa) at 2 mg/mL in PBS, the kit can also be used to label other proteins (>10 kDa).

  2. Add 10 µL of DMSO (Component D) to the vial of reactive dye (Component A), mix well.

  3. Prepare a 500 µL antibody solution in PBS with a concentration of 2 mg/mL.

    Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4. If the protein is dissolved in buffers containing primary amines, like Tris and/or glycine, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, or use Amicon Ultra0.5, Ultracel-10 Membrane, 10 kDa (Cat No. 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) or gelatin will not be labeled well.

  4. Add 25 µL of Reaction Buffer (Component B) to the antibody solution.

  5. Transfer the reconstituted dye solution into the vial of antibody solution, and pipette several times to mix well.

  6. Rotate the reaction mixture for 1 hour at room temperature.

Purification with Desalting Column
  1. Twist off the bottom closure of the desalting column (Component D), and loosen the cap. Place the column in a collection tube.

  2. Centrifuge the column at 1,000 g for 2 minutes to remove the storage solution.

  3. Remove the cap and slowly add 1 mL of PBS to the column. Centrifuge at 1,000 g for 2 minutes and remove the buffer. Repeat this step 3 additional times, discarding the buffer from the collection tube each time.

  4. Place the column in a new collection tube, and gently apply the sample into the center of the compact resin bed.

  5. Centrifuge the column at 1,000 g for 2 minutes to collect the sample.

Determine the Antibody Concentration & Degree of Labeling (Optional)

The following formula can be used to calculate the antibody concentration:

(A280 - CF280 x Adye) / 1.4

The following formula can be used to calculate the degree of labeling:

DOL = (Adye / Ecdye) / (A280 - CF280 x Adye) / 210,000)

Where: 

  • 210,000 is the molar extinction coefficient (Ec) in cm-1M-1 of IgG at 280 nm.
  • CF280 is the correction factor for the effect of the fluorophore on absorbance at 280 nm.
  • Adye is the absorbance at maximum (λmax) for the respective dye.

Table 1. Properties of Labeling Dyes found in the ReadiLink™ Rapid Antibody Labeling Kits.

Cat#

Dye

Mol. Wt.

Ec (cm-1M-1)

CF280

Target DOL

5700

iFluor® 350

749.85

20,000

0.23

5-10

5702

iFluor® 488

945.07

75,000

0.21

4-8

5705

iFluor® 555

914.06

90,000

0.16

4-7

5710

iFluor® 594

1160.42

18,000

0.04

3-6

5713

iFluor® 647

1274.66

250,000

0.03

3-7

5718

iFluor® 750

1416.83

250,000

0.039

2-6

5720

FITC

620.52

75,000

0.183

3-6

5722

Cy3

829.03

150,000

0.073

1-3

5725

Cy5

855.07

250,000

0.03

2-4

5727

Cy7

881.11

250,000

0.036

2-4

5730

XFD488

643.4

71,000

0.11

4-8

5733

XFD555

1250

150,000

0.08

4-7

5736

XFD594

819.85

90,000

0.56

3-6

5740

XFD647

1259.66

240,000

0.03

3-7

5745

XFD750

1300

240,000

0.04

2-5

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*3454502000010.9510.830.23
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*55757010000010.6410.230.14
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*58760320000010.5310.050.04
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*65667025000010.2510.030.03
ReadiLink™ Rapid iFluor® 680 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*68470122000010.2310.0970.094
ReadiLink™ Rapid iFluor® 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*69071322000010.2310.090.04
ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*75777927500010.1210.0440.039
ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*64065425000010.2910.0620.044
ReadiLink™ Rapid iFluor® 790 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*78781225000010.1310.10.09
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit *Production Scale*3454502000010.9510.830.23
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit *Production Scale*55757010000010.6410.230.14
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit *Production Scale*58760320000010.5310.050.04
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Production Scale*65667025000010.2510.030.03
ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit *Production Scale*75777927500010.1210.0440.039
Show More (5)

References

View all 50 references: Citation Explorer
Fluorescence-tagged salivary small extracellular vesicles as a nanotool in early diagnosis of Parkinson's disease.
Authors: Rastogi, Simran and Rani, Komal and Rai, Sanskriti and Singh, Rishabh and Bharti, Prahalad Singh and Sharma, Vaibhav and Sahu, Jyoti and Kapoor, Vrinda and Vishwakarma, Poorvi and Garg, Sumit and Gholap, Shivajirao Lahu and Inampudi, Krishna Kishore and Modi, Gyan Prakash and Rani, Neerja and Tripathi, Madhavi and Srivastava, Achal and Rajan, Roopa and Nikolajeff, Fredrik and Kumar, Saroj
Journal: BMC medicine (2023): 335
Preconcentration of Fluorescent Dyes in Electromembrane Systems via Electrophoretic Migration.
Authors: Kim, Minsung and Kim, Bumjoo
Journal: Micromachines (2023)
Histopathological Investigation of Varietal Responses to Cercospora beticola Infection Process on Sugar Beet Leaves.
Authors: Bhuyian, Md Ziaur Rahman and Solanki, Shyam and Del Rio Mendoza, Luis E and Borowicz, Pawel and Lashman, Dilip and Qi, Aiming and Ameen, Gazala and Khan, Mohamed Fizal
Journal: Plant disease (2023)
Metal-enhanced fluorescence through conventional Ag-polyethylene glycol nanoparticles for cellular imaging.
Authors: Chen, Chih-Jung and Wu, Chun-Yen and Wu, Chi-Wei and Chang, Ching-Wen and Huang, Tsung-Tao and Shiao, Ming-Hua and Lin, Chu-Kuei and Chen, Yu-Chun and Lin, Yung-Sheng
Journal: RSC advances (2023): 26545-26549
Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.
Authors: Okkelman, Irina A and Dmitriev, Ruslan I
Journal: Methods in molecular biology (Clifton, N.J.) (2023): 171-195
Page updated on October 11, 2024

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Catalog Number5702
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Spectral properties

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm -1 M -1)

750001

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.91

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Components