Product Information

Contents: eFluor^TM 605^NC (Amine)
Catalog Number: 93-6366
Sizes: 200 ul
Formulation: 10 μM aqueous solution
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
90-6366	eFluor^TM 490^NC (Amine)	350-500	490	Biomolecule Conjugation
91-6366	eFluor^TM 525^NC (Amine)	350-500	525	Biomolecule Conjugation
93-6366	eFluor^TM 605^NC (Amine)	350-500	605	Biomolecule Conjugation
94-6366	eFluor^TM 625^NC (Amine)	350-500	625	Biomolecule Conjugation
95-6366	eFluor^TM 650^NC (Amine)	350-500	650	Biomolecule Conjugation
96-6366	eFluor^TM 700^NC (Amine)	350-500	700	Biomolecule Conjugation Cell Labeling In vivo Imaging

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Description

Reactive eFluor™ Nanocrystals (Amine), composed of either CdSe or cadmium-free InGaP, are functionalized nanocrystals that can be easily conjugated to proteins, oligonucleotides and other biomolecules of interest using simple binding chemistries and easy-to-complete protocols. The straightforward procedures are designed to allow the user to apply eFluor™ Nanocrystal technology in a wide variety of assay types, such as immunoassays, protein interaction assays, and other fluorescence-based detection assays.

Applications Reported

For research use only, not for diagnostic or therapeutic use. This product has been reported for use in conjugation to biomolecules.

Related Products

Cat. 90-6364  eFluor™ 490NC (Carboxyl)
Cat. 91-6364  eFluor™ 525NC (Carboxyl)
Cat. 93-6364  eFluor™ 605NC (Carboxyl)
Cat. 94-6364  eFluor™ 625NC (Carboxyl)
Cat. 96-6364  eFluor™ 700NC (Carboxyl)
Cat. 90-6366  eFluor™ 490NC (Amine)
Cat. 91-6366  eFluor™ 525NC (Amine)
Cat. 94-6366  eFluor™ 625NC (Amine)
Cat. 96-6366  eFluor™ 700NC (Amine)

Reactive eFluor™ Nanocrystal (Amine) Conjugation Protocol

Materials

2nmol eFluor™ Nanocrystals (Amine) (8-10 uM)
20X Borate Buffer (Pierce, cat. 28341): prepare 1X Borate Buffer in dH2O
BS3, Bis (sulfosuccinimidyl) suberate (Pierce, cat. 21580)
- Prepare 40 mM BS3 solution in 50 mM Borate Buffer, pH8.5 (1X Borate Buffer), just before use.
6-20 nmoles of the biomolecule of interest (equivalent to 1-3 mgs of IgG)
Note: Biomolecule of interest must contain a primary amine group and be free of preservatives, such as BSA and azide. Biomolecule solution must be amine free (1X Borate buffer is recommended) and at high concentration for better coupling efficiency.
Glass vials
PD-10 Desalting column (GE Healthcare, cat. 17-0851-01)
Ultrafiltration units with 100 kDa molecular weight cutoff (Millipore, Amicon Ultra-15, cat. UFC910096)
Note: If biomolecule is <60 kDa use ultrafiltration to separate un-reacted biomolecules from eFluor™ Nanocrystals conjugates.
Superdex 200 (GE Healthcare, cat. 17-1043-10 or cat. 17-5175-01)
Note: If biomolecule is >60 kDa use HPLC or FPLC for optimal separation of un-reacted biomolecules from eFluor™ Nanocrystals conjugates.

Method

eFluor™ Nanocrystal (Amine) Activation
1. Transfer 2.0 nmoles of eFluor™ Nanocrystals (Amine) to a glass vial and activate by adding 50uL of 40mM BS3 dissolved in 50mM Borate buffer (pH 8.5).
2. Incubate the reaction mixture for 30 minutes at room temperature while gently mixing.
3. During the incubation period, equilibrate a PD-10 Desalting column with 1X Borate buffer according to manufacturers instructions.
4. Load the reaction mixture onto the PD-10 Desalting column and allow the sample to enter the gel bed completely. Elute the activated eFluor™ Nanocrystals (Amine) into a glass vial by adding 3-5 mls of 1X Borate buffer to the column.
  Note: Collect only the fluorescent colored fraction. To avoid sample contamination with excess cross-linker (BS3) do not collect any sample after the fluorescent colored fraction (use a black light to monitor elution).
5. Concentrate the purified activated eFluor™ Nanocrystals (Amine) to 200ul using Ultrafiltration unit with 100 kDa molecular weight cutoff
Biomolecule Coupling
1. Add 6-20 nmoles of the biomolecule of interest to the purified activated eFluor™ Nanocrystals (Amine).
  Note: The stoichiometry of the reaction must be optimized to ensure maximum coupling efficiency.
2. Incubate the reaction at room temperature for 1.25 hours while gently mixing. Protect reaction from light.
Conjugate Purification
1. Biomolecules < 60kDa:
  Add the conjugate to an Ultrafiltration unit with 100 kDa molecular weight cutoff and wash 5-6 times with 1X Borate buffer. Concentrate conjugate to <500µl.
  
  Biomolecules >60Kda:
  Purify the conjugate by FPLC or HPLC size exclusion chromatograpy using a Superdex 200 matrix. Pool the fluorescent fraction (use a black light to identify fluorescent fractions), and concentrate to < 500ul using an Ultrafiltration unit with 100 kDa molecular weight cutoff. Pool the fractions containing free biomolecule and quantify the amount of un-reacted material to determine the coupling efficiency.