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ReadiLink™ Rapid FITC 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 FITC 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. FITC is a green fluorescent dye with an excitation maximum of ~491 nm and an emission maximum of ~516 nm. It has a relatively high rate of photobleaching and a fluorescence signal sensitive to pH changes.

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 Cy3 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*55556915000010.1510.070.073
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*65167025000010.271, 0.420.020.03
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*7567792500000.30.050.036
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *XFD488 Same Structure to Alexa Fluor™ 488*499520710000.9210.300.11
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *XFD555 Same Structure to Alexa Fluor™ 555*5535681500000.110.080.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *XFD594 Same Structure to Alexa Fluor™ 594*590618900000.6610.430.56
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *XFD647 Same Structure to Alexa Fluor™ 647*6506712390000.3310.000.03
ReadiLink™ Rapid XFD750 Antibody Labeling Kit *XFD750 Same Structure to Alexa Fluor™ 750*7527762400000.1210.000.04
ReadiLink™ Rapid Cy3 Antibody Labeling Kit *Production Scale*55556915000010.1510.070.073
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Production Scale*65167025000010.271, 0.420.020.03
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *Production Scale*499520710000.9210.300.11
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *Production Scale*5535681500000.110.080.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *Production Scale*590618900000.6610.430.56
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Production Scale*7567792500000.30.050.036
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *Production Scale*6506712390000.3310.000.03
ReadiLink™ Rapid XFD750 Antibody Labeling Kit *Production Scale*7527762400000.1210.000.04
Show More (7)

References

View all 50 references: Citation Explorer
Characterization and optimization of fluorescein isothiocyanate labeling of humanized h2E2 anti-cocaine mAb.
Authors: Kirley, Terence L and Norman, Andrew B
Journal: Biochemistry and biophysics reports (2023): 101520
Evans Blue and Fluorescein Isothiocyanate-Dextran Double Labeling Reveals Precise Sequence of Vascular Leakage and Glial Responses After Exposure to Mild-Level Blast-Associated Shock Waves.
Authors: Nishii, Kiyomasa and Satoh, Yasushi and Higashi, Takahito and Matsui, Toshiyasu and Ishizuka, Toshiaki and Kashitani, Masashi and Saitoh, Daizoh and Kobayashi, Yasushi
Journal: Journal of neurotrauma (2023): 1228-1242
Estimating Changes in Glomerular Filtration Rate With Fluorescein Isothiocyanate-Sinistrin During Renal Replacement Therapy.
Authors: Chan, Goldia and Pino, Christopher J and Johnston, Kimberly A and Humes, H David
Journal: ASAIO journal (American Society for Artificial Internal Organs : 1992) (2023): 810-815
FRET causing misleading signal from fluorescein excited by the violet laser in flow cytometry.
Authors: Waeckel, Louis and Khenine, Hana and Berger, Anne-Emmanuelle and Lambert, Claude
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2023)
A proof-of-concept study on the use of a fluorescein-based 18F-tracer for pretargeted PET.
Authors: Helbert, Hugo and Ploeg, Emily M and Samplonius, Douwe F and Blok, Simon N and Antunes, Ines F and Böhmer, Verena I and Luurtsema, Gert and Dierckx, Rudi A J O and Feringa, Ben L and Elsinga, Philip H and Szymanski, Wiktor and Helfrich, Wijnand
Journal: EJNMMI radiopharmacy and chemistry (2022): 3
Page updated on October 9, 2024

Ordering information

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Catalog Number5720
Quantity
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Spectral properties

Correction Factor (280 nm)

0.35

Extinction coefficient (cm -1 M -1)

73000

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.92

Storage, safety and handling

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

Components