Thermo Fisher Scientific DyLight Long Stokes Shift Dyes User Guide
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INSTRUCTIONS
DyLight
®
Long Stokes Shift Dyes
82491 82492 82493 82495
Number Description
82492 DyLight 485-LS NHS Ester, 1mg
2448.0
82493
82491
82495
DyLight 510-LS NHS Ester, 1mg
DyLight 515-LS NHS Ester, 1mg
DyLight 521-LS NHS Ester, 1mg
Storage: Upon receipt store at -20
°
C. Product shipped at ambient temperature.
Introduction
The Thermo Scientific DyLight Long Stokes Shift Dyes are amine-reactive fluorescent dyes that produce long Stokes (LS) shifts of 80-145nm in excitation/emission wavelengths (see Table 1). The DyLight LS Dyes provide multicolor detection using a single excitation source in applications including flow cytometry, conjugation, DNA-sequencing, microscopy and fluorescence in situ hybridization (FISH).
These dyes contain N -hydroxysuccinimide (NHS) esters, the most commonly used reactive group for labeling proteins. NHS esters react with primary amines on proteins and other molecules, forming a stable, covalent amide bond and releasing the
NHS group.
Table 1. Properties of the Thermo Scientific DyLight LS Dyes using a 488nm excitation laser line.
DyLight
Dye
485-LS
Ex/Em*
485/559
ε †
50,000
MW
(g/mol)
600
Emission
Laser Line
Yellow
510-LS
515-LS
509/590
515/650
50,000
50,000
652
728
Orange
Red
521-LS 523/668 50,000 728 Far Red
*
Excitation and emission maxima in nanometers
†
Molar extinction coefficient (M
-1 cm
-1
)
Important Product Information
•
NHS ester-activated fluorophores are moisture-sensitive. Store product in the original pouch at -20
°
C. Avoid moisture condensation onto the product by equilibrating the vial to room temperature before opening. Prepare the labeling reagents immediately before use. Do not store NHS-ester reagents in aqueous solutions.
•
Low concentrations of sodium azide (
≤
3mM or 0.02%) or thimerosal (
≤
0.02mM or 0.01%) will not significantly interfere with protein labeling; however, 20-50% glycerol will reduce labeling efficiency.
•
To remove excess non-reacted dye, use a dialysis membrane with a molecularweight cutoff ≥10K or the Thermo
Scientific Pierce Dye Removal Columns (Product No. 22858).
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
(815) 968-0747
(815) 968-7316 fax www.thermoscientific.com/pierce
Procedure for Protein Labeling
Note: The following protocol is an example labeling application; specific applications require optimization.
A.
Protein Preparation
The optimal labeling buffer is 0.05M sodium borate buffer at pH 8.5 (e.g., Thermo Scientific BupH Borate Buffer Packs,
Product No. 28384). Buffers containing primary amines (e.g., Tris or glycine) will interfere because they react with the NHSester moiety. Dissolve protein directly in the labeling buffer. For each labeling reaction, use
100μL to 1mL of purified protein sample at 1-10mg/mL. After reconstitution, proceed to the Calculations for Labeling Section. If the protein is already in a buffer, perform a buffer exchange into the labeling buffer by dialysis or gel filtration.
Note: The following buffers may be substituted for borate buffer: 0.1M sodium phosphate, 0.15M NaCl at pH 7.2-7.5 (e.g.,
BupH™ Phosphate Buffered Saline Packs, Product No. 28372) OR 0.1M sodium carbonate at pH 8.3-9.0.
B.
DyLight Dye Preparation
Equilibrate vial to room temperature before opening to avoid moisture condensation onto the reagent. Dissolve reagent in
DMF at 10mg/mL. The reagent may also be dissolved at 1mg/mL to make pipetting small amounts more accurate; however, adjust for the concentration change when calculating the reagent amount added to the labeling reaction.
C.
Calculations for Labeling
The amount of fluorescent-labeling reagent to use for each reaction depends on the amount of protein to be labeled and the specific fluorophore being used. Generally, the more concentrated the protein, the more efficient the reaction.
1.
Calculate the amount (mg) of DyLight LS NHS-Ester Dye to be added to the labeling reaction: amount of protein (mg)
×
10
×
MW of dye
MW of protein
=
_______ mg of dye
•
10 = Molar-fold excess of the NHS ester dye to protein
2.
Calculate microliters of dye solution to add to the reaction: mg of dye (calculati on #1)
×
100
μL
1 mg
=
_______
μL
NHS ester dye solution at 10 mg/mL
• 100μL = Solvent volume in which the 1mg of NHS-ester dye is dissolved
Example Calculation:
For 1mL of a 2mg/mL solution of IgG (150,000 MW), 9.7μL of DyLight 515- LS NHS
Ester (10mg/mL) will be used.
0.097
mg of
2 mg IgG
150,000 MW
×
10
×
727.75
=
0.097
mg of DyLight 515
DyLight 515 LS NHS Ester
×
100
μL
1 mg
=
9.7
μL of
LS NHS Ester
DyLight 515 LS NHS Ester
D.
Labeling Reaction
1.
Equilibrate dyes to room temperature before opening vials.
2.
Add 100μL of DMF to the dye. Pipette up and down or vortex until it is completely dissolved.
Note: Allow the dye to completely dissolve for 5-10 minutes and then vortex again.
3.
Transfer the protein solution to be labeled to a reaction tube.
4.
Add the calculated amount of reagent to the reaction tube containing the protein. Mix well and incubate at room temperature for 1 hour, protected from light.
5.
Remove non-reacted reagent from the protein by dialysis or with Pierce
®
Dye Removal Columns.
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
2
(815) 968-0747
(815) 968-7316 fax www.thermoscientific.com/pierce
6.
Store labeled protein protected from light at 4
°
C for up to one month.
Note: For long-term storage, add bovine serum albumin (5-10mg/mL) and sodium azide (0.01-0.03% final concentration) to the conjugate and store the labeled protein in single-use volumes at -20
°
C. Exact storage conditions may vary for different proteins and should be determined empirically.
E.
Calculate the Degree of Labeling
1.
Remove excess dye reagent from the sample using a dialysis membrane with a molecular-weight cutoff
≥
10K or the
Pierce Dye Removal Columns.
Note: For optimal results and accurate determination of the dye-to-protein ratio, remove all non-reacted dye. Remove excess dye by dialyzing for ~4 hours using three dialysis buffer changes. Gel filtration (e.g., desalting columns) is not as effective as dialysis and, therefore, is not recommended.
2.
Dilute a small amount of labeled, purified protein in PBS. Using a 1cm path length cuvette, measure the absorbance at
280nm (A
280
) and the A max
of the specific dye (Table 2).
Table 2. Properties of the Thermo Scientific DyLight LS Dyes.
DyLight Dye
485-LS
A max
*
485
ε †
50,000
CF
§
0.137 (in EtOH)
510-LS 509 50,000 0.174 (in EtOH)
515-LS 519 50,000
0.142 (in EtOH)
0.105 (in PBS)
521-LS 526 50,000
0.184 (in EtOH)
0.155 (in PBS)
*
Excitation wavelength – note that upon protein conjugation, the absorption maximum shifts to the right of the spectra
†
Molar extinction coefficient (M
-1 cm
-1
) at A max
§
Correction factor (A
280
/A max
) in ethanol (EtOH) or phosphatebuffered saline (PBS)
3.
Calculate protein concentration as follows:
Protein concentrat ion (M)
=
[A
280
(A max
ε protein
×
CF)]
× dilution factor
• ε protein
= protein molar extinction coefficient (e.g., the molar extinction coefficient of IgG is ~210,000 M
-1
cm
-1
)
•
CF = Correction factor
=
A
280
A max of of
the dye
the dye
(see Table 2)
4.
Calculate the degree of labeling:
Moles of dye per mole of protein
=
A max of
ε
the dye
× labeled protein protein
× concentrat dilution ion (M) factor
• ε dye
= see Table 2
Example calculations for DyLight 515-LS Dye conjugated to antibodies in PBS:
Dilution factor = 10
A
280
= 0.287
A max
at 519nm = 0.878
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
3
(815) 968-0747
(815) 968-7316 fax www.thermoscientific.com/pierce
Protein concentration calculation:
Protein concentrat ion (M)
=
[0.287
(0.878
×
0.105)]
×
10
210,000
=
0.0000093
M
Mole Dye-to-mole protein calculation:
Moles of dye per mole of protein
=
0.878
150,000
×
×
10
0.0000093
=
6.3
Troubleshooting
Problem
Dye-labeled protein application is unsuccessful
Cause
The protein was not labeled
Solution
Before troubleshooting, determine if the protein is labeled by calculating the A max
:A
280
ratio; determine this ratio after thorough desalting or dialysis
Note: For dye-labeled antibodies the A max should be
>
1.
:A
280
ratio
Use a conjugation buffer (e.g., borate, carbonate, PBS) free of primary amines
Protein is not labeled Conjugation Buffer contained primary amines (e.g., Tris or glycine) that interfered with the reaction
The NHS ester hydrolyzed, becoming non-reactive
Additional Information
Prepare labeling reagent immediately before use
−
do not store NHS-ester reagents in aqueous solutions
Visit our website for additional information including the following items:
•
Tech Tip #43: Protein stability and storage
•
Tech Tip #3: Determine reactivity of NHS-ester biotinylation and crosslinking reagents
•
Tech Tip #30: Modify and label oligonucleotide 5´ phosphate groups
Related Thermo Scientific Products
22858 Pierce Dye Removal Columns
28384
28341
28372
28348
BupH Borate Buffer Packs,
20X Borate Buffer, 500mL
40/pkg
BupH Phosphate Buffered Saline Packs,
20X Phosphate Buffered Saline (PBS),
40/pkg
500mL
This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the
Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).
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Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
4
(815) 968-0747
(815) 968-7316 fax www.thermoscientific.com/pierce

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