Product manual
Product use, preparation, operating conditions and storage.
SF594Reactive dye
SF594-NH2
Cat. No.QH-12-1010(NH2)
SF–NH2 supplies a free amine for SMCC activation followed by target-thiol coupling, or for a suitable carboxyl-activation route.
Packs & quote1 · Install maleimide; remove free SMCC
NH₂
Dye amineSMCC
NHS / MaleimideMal
Dye–maleimide intermediate2 · Couple target free thiols
Mal
Purified intermediateR–SH
Target free thiolS–R
Thioether linkageExcitation590 nm
Emission617 nm
TargetActivated carboxyls, or free thiols through a linker such as SMCC
Preparation and storage
- Preparation
- SF–NH2 can be prepared in aqueous PBS and activated with SMCC for target-thiol coupling. Confirm buffer composition, concentration and intermediate purification for the selected procedure.
- Storage
- Store at −20°C, dry and protected from light; avoid repeated freeze–thaw cycles.
Free amine and sequential coupling
NH2 is a primary amine on the dye, not an NHS ester. Two unactivated primary amines do not couple directly. Use carboxyl activation, or react the dye amine with the NHS end of SMCC before coupling its maleimide end to a target thiol.
Activated-carboxyl samples
For molecules or materials with suitable activated carboxyl groups. Control activation and coupling separately and assess sample self-crosslinking.
Sequential protein labeling
Connect the dye amine to a target thiol through a linker, removing excess linker and free dye between appropriate steps.
SF-conjugate assessment
Assess positive signal, negative background, recovery and function of the final conjugate. Flow-cytometric gate fractions, fluorescence intensity and bleaching under continuous illumination are different endpoints and should be recorded separately.
Specifications
Product information
- Product name
- SF594-NH2
- Cat. No.
- QH-12-1010(NH2)
- Product form
- NH2
- Target
- Activated carboxyls, or free thiols through a linker such as SMCC
- Pack sizes
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
Preparation and properties
- Excitation wavelength
- 590 nm
- Emission wavelength
- 617 nm
- Preparation and sample
- SF–NH2 can be prepared in aqueous PBS and activated with SMCC for target-thiol coupling. Confirm buffer composition, concentration and intermediate purification for the selected procedure.
- Storage
- Store at −20°C, dry and protected from light; avoid repeated freeze–thaw cycles.
Spectral information and detection
Product parameters
Use parameters for the exact catalog number when preparing reagents, setting reagent input or calculating results. Request a lot-specific CoA for test results.
Protocol
Confirm both functional groups
The dye carries NH2; the target needs an activated carboxyl or a group matched to the linker. Do not add it to a native antibody as though it were an NHS dye.
Plan intermediate purification
Define the buffer and separation method for each step to limit linker reactions with unintended sample components.
Aqueous preparation of the NH2 form
The SF amine-labeling procedure uses 5–10 mg/mL SF in 0.1 M PBS, pH 7.4, with 5 mM EDTA. This condition belongs to the NH2 route and does not establish the solvent or concentration for SF–DBCO.
Prepare target free thiols
Assess sample thiols first. If reduction is needed, control its extent and remove competing components such as DTT and β-mercaptoethanol before coupling. Retain an unlabeled aliquot to assess integrity.
Activate SF–NH2 with SMCC
React the SF amine with the NHS end of SMCC to install maleimide. A reference procedure uses a 10 mg/mL SMCC stock in anhydrous DMSO and about one hour of activation at room temperature, protected from light. Set input from accessible amines and sample requirements.
Remove linker before coupling
Remove free SMCC using a separation that retains the activated SF intermediate, then couple to target thiols. Excess SMCC can modify unintended protein sites; use the purified intermediate promptly.
Purification and analysis
Select desalting, size-exclusion separation, dialysis or chromatography for the sample size. Remove free reagent before measuring concentration, labeling density and function. Residual free dye raises background and biases labeling-density calculations.
Check coupling and function
Compare purified products with and without SMCC treatment, recording recovery, aggregation, specific signal and function. If remaining thiols require blocking, follow the blocker’s instructions and remove small molecules again.
Request product documentation
Select a document and enter your phone and email. Include the lot number for a CoA.
SF594-NH2QH-12-1010(NH2)
Spectra and measurement conditions
Spectra, measurement conditions and detection settings for the exact catalog number.
Certificate of analysis (CoA)
Lot-specific test results. Include the lot number from the vial.
Safety data sheet (SDS)
Handling, storage and emergency response information.
Quality and lot information
No-activation control
Omit activator or linker, purify identically and assess adsorption background.
Unlabeled sample
Use an unlabeled aliquot from the same preparation to assess changes in function, aggregation and recovery.
Free dye and negative sample
Include free-dye or no-target-handle controls to distinguish conjugation from adsorption. Match exposure, gain or voltage during detection.
Technical articles
Application support
Include your sample, formulation and detection method when requesting application guidance.
References
Stepwise amine-to-thiol coupling with SMCC
Thermo Fisher Scientific · SMCC and Sulfo-SMCC · MAN0011295 Rev. C.0
Distinguishes the partners and reaction order of the NHS and maleimide ends and removal of free linker, supporting bridging routes for NH2 dyes.
Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with maleimide and alpha-haloacyl groups: anomalous elution of TCEP by gel filtration
Shafer DE, Inman JK, Lees A. · Analytical Biochemistry · 282(1):161–164 · 2000
Examines TCEP interactions with reactive groups, informing evaluation of reductant carryover and buffer exchange.
Frequently asked questions
Are NH2 and NHS the same type of product?
No. NH2 supplies a free primary amine; NHS supplies an activated ester that reacts with an amine. Their partners and workflows differ.
Can it react directly with antibody lysines?
Two ordinary primary amines do not couple directly. Use an appropriate activation or bridging route.
Can intermediate purification be skipped?
This depends on the reaction design. Residual linker can consume target sites or cause self-crosslinking; validate before simplifying the workflow.