BIOTIUM CF Reactive Dyes and Protein Labelling Kits Owner's Manual

Biotium CF Reactive Dyes and Protein Labelling Kits offer versatile tools for fluorescent protein labeling. These kits utilize carbodiimide chemistry (EDC coupling) for direct conjugation or a two-stage EDC/Sulfo-NHS method for enhanced efficiency. Various CF® dyes with distinct excitation/emission wavelengths are available, enabling multicolor applications. The kits include protocols for both one-step and two-step labeling, allowing flexibility depending on your protein and experimental needs. Purification methods using Sephadex columns or ultrafiltration are described to remove excess dye.

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Product Information
CF&reg; Dye Amine
Revised: June 29, 2020
Experimental Protocols
Direct EDC Coupling Protocol for
Labeling Proteins with CF&reg; Dye Amines
Unit Size: 1 mg
Extinction
Catalog no.
Dye
Ex/Em (nm)
92035
CF&reg;350
347/448
18,000
~537
92036
CF&reg;405S
408/452
33,000
~725
92037
CF&reg;488A
490/515
70,000
~704
92038
CF&reg;555
555/565
150,000
~943
92039
CF&reg;568
562/583
100,000
~756
~771
coefficient
MW
EDC is a popular carbodiimide for conjugating carboxylate groups to primary
amines between biological substances.1 EDC reacts with carboxylic acids to form
a highly reactive, O-acylisourea intermediate. This intermediate can then react with
a nucleophilic primary amine to form an amide bond. Please note that some side
reactions may occur when using EDC with proteins. For instance, EDC can form a
stable complex with exposed sulfhydryl groups and tyrosine residues.2-3 In addition,
EDC may promote unwanted polymerization due to the presence of both amines
and carboxylates on protein molecules. The following protocol has been adapted
from literature for conjugating CF&reg; dye amines to proteins using EDC coupling.1,4,5
The protocol may be modified by changing the pH, buffer salts, and ratios of
reactants to obtain the desired product.
92040
CF&reg;594
593/614
115,000
92041
CF&reg;633
630/650
100,000
~863
92043
CF&reg;640R
642/662
105,000
~1034
92042
CF&reg;647
650/665
240,000
~1027
96010
CF&reg;660R
663/682
100,000
~930
• EDC (EDAC) (catalog no. 59002)
92102
CF&reg;750
755/777
250,000
~3005
• PBS buffer (pH 7.4) (catalog no. 22020)
92065
CF&reg;770
770/797
250,000
~3134
• (Optional) Ultrafiltration Vial (see related products)
Storage and Handling
Store CF&reg; dye amine at -20&deg;C, protected from light. Product is stable for at least
12 months from date of receipt if stored as recommended. Stock solutions may be
prepared in DMSO or dH2O and can be stored at ≤ -20&deg;C for at least 12 months.
Materials required but not provided
• Anhydrous DMSO (see related products)
• Reaction Buffer: 0.1 M MES (2-[N-morpholino]ethanesulfonic acid), pH 4.7-6
• Protein sample in Reaction Buffer
• Sephadex&reg;; see Table 1 for the appropriate type of Sephadex&reg; for each
CF&reg; dye
One-Step Labeling Protocol
1.
Equilibrate EDC to room temperature.
Product Description
CF&reg; dye amines can be conjugated to activated carboxylic acids in proteins
or other molecules using carbodiimide chemistry. EDC (EDAC) (catalog
no. 59002) may be used for direct coupling of carboxylic acids to primary
amines using carbodiimide chemistry. Alternatively, EDC may be coupled with
N-Hydroxysuccinimide (NHS) to prepare semi-stable NHS esters from carboxylate
groups that can then be conjugated to primary amines. Our CF&reg; amine derivatives
are bright, photostable and water-soluble, making them an excellent choice for
fluorescent labeling.
2.
Dissolve the protein to be modified in 200 uL of Reaction Buffer for a final
concentration of 20-100 uM.
3.
Add CF&reg; Dye Amine stock solution to protein solution in 10-fold molar
excess. For example, 200 uL of 50 uM protein in reaction buffer is 10 nmole
of protein total. Therefore add 100 nmole of CF&reg; Dye Amine (or 20 uL of
5 mM stock solution).
References
1) Hermanson, G. (1996). Bioconjugate Techniques (1st ed.); 2) Biochim. Biophys.
200(3), 546(1970); 3) Biochim. Biophys. 160(2). 272(1968); 4) Anal. Biochem.
185(1), 131(1990); 5) Anal. Biochem. 156(1), 220(1986); 6) Nanotheranostics.
2(4), 347(2018); 7) Proc Natl Acad Sci USA. 116(20), 9831(2019).
4.
Add EDC to the reaction to obtain at least a 10-fold molar excess of EDC to
the protein. Mix reaction well.
Note: Water or 0.1 M sodium phosphate, pH 7.3 may also be used. NaCl
may also be added to the buffer if desired.
Note: 0.5-0.1 M EDC in the reaction is usually a suitable concentration. For
convenience, the reaction solution may be added to a tube containing 10 mg
of EDC. If precipitation occurs, reduce the amount of EDC until the conjugate
is soluble.
5.
Incubate reaction for at least 2 hours at room temperature in the dark.
6.
Separate the labeled protein from the free dye.
a. Prepare a Sephadex&reg; column (10 mm x 300 mm) equilibrated in PBS
buffer (pH~7.4).
b. After incubation, load the reaction solution onto the column and elute
the column with PBS buffer. The first band excluded from the column
corresponds to the antibody conjugate.
Note: See Table 1 for the appropriate Sephadex&reg; medium to use for each
CF&reg; dye. For small scale labeling reactions, you may use an ultrafiltration
vial (see related products) to remove the free dye from the conjugate in
order to avoid an overly dilute product. Choose an ultrafiltration vial with a
molecular weight cut-off at least 3X small than the protein molecular weight.
CF&reg; Dye AminePage 1 of 3
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7.
Store conjugate in an appropriate buffer and temperature for the protein of
interest, protected from light.
9.
Separate the labeled protein from the free dye.
a. Prepare a Sephadex&reg; column (10 mm x 300 mm) equilibrated in PBS
buffer (pH~7.4).
Two-Stage EDC/Sulfo-NHS Coupling Protocol for
Labeling Proteins with CF&reg; Dye Amines
The following protocol is a modified two-step protocol which involves activation
of carboxyl proteins with EDC/sulfo-NHS and subsequent conjugation with CF&reg;
dye amines.1,4 Sulfo-NHS improves the EDC coupling efficiency by increasing
the stability of the O-acylisourea intermediate, thereby extending the half-life
of the activated carboxylate to hours. The protocol involves an acidic pH of
activation which provides greater stability for the active ester intermediate.
2-mercaptoethanol is also used to quench any unreacted EDC. The protocol may
be modified by changing the pH, buffer salts, and ratios of reactants to obtain the
desired product.
Materials required but not provided
• Anhydrous DMSO (see related products)
• Reaction Buffer: 0.05 M MES (2-[N-morpholino]ethanesulfonic acid), 0.5 M
NaCl, pH 6
b. After incubation, load the reaction solution onto the column and elute
the column with PBS buffer. The first band eluted from the column
corresponds to the protein conjugate.
Note: See Table 1 for the appropriate Sephadex&reg; medium to use for each
CF&reg; dye. For small scale labeling reactions, you may use an ultrafiltration
vial (see related products) to remove the free dye from the conjugate in
order to avoid an overly dilute product. Choose an ultrafiltration vial with
a molecular weight cut-off at least 3X smaller than the protein molecular
weight.
10.
Store conjugate in an appropriate buffer and temperature for the protein of
interest, protected from light.
Table 1. CF&reg; Dye Technical Data
Dye
Sephadex&reg;
Amax (nm)
media
A280/Amax or
Cf (protein)
Extinction
coefficient (ε)
Optimal
DOL (IgG)
• Protein sample in Reaction Buffer
CF&reg;350
G-25
347
0.14
18,000
4-6
• EDC (EDAC) (catalog no. 59002)
CF&reg;405S
G-25
404
0.7
33,000
5-10
• Sulfo-NHS (N-Hydroxysuccinimide)
CF&reg;405M
G-25
408
0.13
41,000
4-6
• 2-mercaptoethanol
CF&reg;405L
G-25
395
0.5
24,000
8-12
CF&reg;430
G-25
426
0.044
40,000
5-8
CF&reg;440
G-25
440
0.044
40,000
5-8
CF&reg;450
G-25
450
0.2
40,000
5-8
• PBS buffer (pH 7.4) (catalog no. 22020)
• (Optional) Hydroxylamine
• (Optional) Ultrafiltration Vial (see related products)
• (Optional) Sephadex&reg; G-25 desalting column
• Sephadex&reg;; see Table 1 for the appropriate type of Sephadex&reg; for each
CF&reg; dye
Two-Step Labeling Protocol
1.
Equilibrate EDC to room temperature.
2.
Dissolve the protein to be modified in 200 uL of Reaction Buffer for a final
concentration of 20-100 uM.
3.
Add EDC and sulfo-NHS to the solution for a final concentration of 2 mM EDC and 5 mM sulfo-NHS. Mix reaction well.
CF&reg;488A
G-25
490
0.1
70,000
7-9
CF&reg;503R
G-25
503
0.09
90,000
4-10
CF&reg;514
G-25
516
0.073
105,000
5-8
CF&reg;532
G-25
527
0.06
96,000
4-7
CF&reg;543
G-25
541
0.095
100,000
4-7
CF&reg;550R
G-25
551
0.08
100,000
5-6
CF&reg;555
G-25
555
0.08
150,000
4-5, 3-6 ok
0.08
100,000
5-6
CF&reg;568
G-25
562
Note: To achieve accurate final concentrations, EDC and sulfo-NHS may
be quickly dissolved in reaction buffer at higher concentrations, and then
immediately pipetted into the protein solution to achieve the appropriate final
concentrations.
CF&reg;570
G-25
568
0.1
150,000
5-6
CF&reg;583
G-25
583
0.223
150,000
5-6
CF&reg;594
G-25
593
0.08
115,000
4-7
3.
Allow reaction to incubate for 15 minutes at room temperature.
CF&reg;620R
G-25
617
0.45
115,000
5-6
4.
Add 2-mercaptoethanol to the a reaction solution for a final concentration of
20 mM. Mix well and incubate at room temperature for 10 minutes.
CF&reg;633
G-25
630
0.48
100,000
4-7
CF&reg;640R
G-50
642
0.37
105,000
4-7
CF&reg;647
G-25
650
0.03
240,000
4-5, 3-6 ok
Optional: Use a Sephadex&reg; G-25 desalting column or equivalent to purify
the activated protein.
CF&reg;660C
G-75
667
0.08
200,000
3-6, 2-3 ok
CF&reg;660R
G-25
663
0.51
100,000
4-7
Note: The desalting process should be done rapidly to minimize hydrolysis
and recover as much of the active ester as possible.
CF&reg;680
G-75
681
0.09
210,000
3-5, 2-3 ok
CF&reg;680R
G-25
680
0.32
140,000
5-6
CF&reg;700
G-75
695
0.06
240,000
3-6
CF&reg;750
G-75
755
0.03
250,000
3-5, 2-3 ok
CF&reg;770
G-75
770
0.06
220,000
3-5, 2-3 ok
Note: The increase in pH above 7.0 is require to initiate the active ester
reaction.
CF&reg;790
G-75
784
0.07
210,000
3-5
7.
Incubate reaction for at least 2 hours at room temperature in the dark.
CF&reg;800
G-75
797
0.08
210,000
3-5
8.
Optional: Quench the reaction by adding hydroxylamine to a final concentration of 10 mM. Mix reaction well.
CF&reg;820
G-75
822
0.07
253,000
3-6
Note: If the protein is sensitive to 2-mercaptoethanol, the activation may also
be terminated by desalting (step 5)
5.
6.
Add 10-fold molar excess of CF&reg; Dye Amine dissolved in concentrated PBS
(catalog no. 22020) or other non-amine buffer to increase pH above 7.0. For
example, 200 uL of 50 uM protein in reaction buffer is 10 nmole of protein
total. Therefore add 100 nmole of CF&reg; Dye Amine (or 20 uL of 5 mM stock
solution). Mix reaction well.
Note: Alternative quenching reagents include 20-50 mM Tris, lysine,
glycine and ethanolamine.
CF&reg; Dye AminePage 2 of 3
PSF006
Related Products
Catalog
number
22004
22018
90082
59002
22013
22014
22020
41024-4L
Product
Ultrafiltration Vial, 10K MWCO (5 per pack)
Ultrafiltration Vial, 3K MWCO (5 per pack)
DMSO, Anhydrous, 10 mL
EDC (EDAC), 100 mg
Bovine Serum Albumin, Fraction V, 50 g
Bovine Serum Albumin, 30% Solution, 100 mL
10X Phosphate Buffered Saline, 4 L
Water, Ultrapure Molecular Biology Grade, 4 L
Please visit www.biotium.com to view our full selection of CF&reg; reactive dyes and
labeling kits, CF&reg; dye labeled antibodies and other conjugates, and more.
CF dye technology is covered by pending US and international patents. Sephadex
are registered trademarks of GE Healthcare. Materials from Biotium are sold for
research use only, and are not intended for food, drug, household, or cosmetic use.
CF&reg; Dye AminePage 3 of 3
PSF006
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Key features

Multiple CF® dyes
One-step & two-step protocols
High labeling efficiency
Photostable dyes
Water-soluble dyes

Frequently asked questions

The viewed document describes labeling proteins with activated carboxylic acids using carbodiimide chemistry. Specific proteins mentioned are antibodies, but the method adapts to other suitable proteins.

According to the viewed document, EDC (or EDAC) is a carbodiimide used for conjugating carboxylate groups to primary amines in proteins. It forms a reactive intermediate that facilitates the amide bond formation between the dye and the protein.

The viewed document suggests using a Sephadex® column (specific type depending on the dye used) or ultrafiltration with a molecular weight cut-off at least 3 times smaller than the protein's molecular weight.

The viewed document states that CF® dye amines should be stored at -20°C, protected from light. Stock solutions in DMSO or dH2O can be stored at ≤ -20°C.

Yes, the viewed document provides protocols for both one-step direct EDC coupling and a two-step EDC/Sulfo-NHS coupling protocol.