5-FAM Hydrazide HCl: Properties, Uses & FAQs

5-FAM hydrazide HCl is a carbonyl-reactive, green-fluorescent labeling reagent that enables selective covalent conjugation of the fluorescein fluorophore to aldehydes, ketones, and oxidized carbohydrates under mild aqueous conditions.

SKU

F595960

Fluorophore

5-Carboxyfluorescein (5-FAM)

Reactive Group

Hydrazide (–CONHNH₂), reacts with aldehydes and ketones to form hydrazone bonds

Excitation / Emission

Approximately 492 nm excitation / 517 nm emission (fluorescein-compatible filter sets)

Solubility

Soluble in water and DMSO; prepare stock solutions in DMSO or aqueous buffer at mildly acidic to neutral pH

Storage

Store at −20 °C, protected from light and moisture; avoid repeated freeze-thaw cycles

Primary Applications

Glycan labeling, oxidized biomolecule detection, carbonyl-group bioconjugation, flow cytometry, fluorescence microscopy

What Is 5-FAM Hydrazide HCl?

5-FAM hydrazide HCl (Aladdin SKU F595960) is the hydrochloride salt of 5-carboxyfluorescein hydrazide, a carbonyl-selective fluorescent probe based on the well-characterized fluorescein scaffold. The hydrazide functional group (–CONHNH₂) reacts selectively and efficiently with aldehyde (–CHO) and ketone (–C=O) groups to form stable hydrazone (–C=N–NH–) linkages under mild, near-physiological conditions.

Because it carries the 5-FAM fluorophore, conjugates are detectable using standard FITC/fluorescein optical configurations (excitation ~492 nm, emission ~517 nm), making the reagent compatible with the vast majority of fluorescence microscopes, plate readers, and flow cytometers found in modern life-science laboratories.

Key Applications in Research

Glycan and Carbohydrate Labeling: Polysaccharides and glycoproteins can be oxidized with periodate (NaIO₄) to generate aldehyde groups on vicinal diols. 5-FAM hydrazide then reacts with these aldehydes to yield fluorescently tagged glycoconjugates suitable for flow cytometry, confocal microscopy, or gel-based detection.

Oxidized Biomolecule Detection: Reactive oxygen species (ROS) can oxidize lipids and proteins to introduce carbonyl groups. 5-FAM hydrazide provides a fluorescent readout of oxidative damage, complementing carbonylation assays that traditionally use dinitrophenylhydrazine (DNPH).

General Bioconjugation: Any target molecule bearing an aldehyde or ketone handle — including small molecules, peptides, proteins, oligonucleotides, or surfaces — can be fluorescently labeled site-selectively using this reagent, enabling tracking, quantification, and imaging studies.

Handling, Solubility & Storage Guidelines

Solubility: 5-FAM hydrazide HCl is soluble in water and in DMSO. For aqueous applications, dissolve directly in PBS or a mildly acidic buffer (pH 5–7). DMSO stock solutions (e.g., 10–50 mM) can be prepared and diluted into the reaction buffer immediately before use. Avoid strongly alkaline conditions that may hydrolyze the hydrazide or quench fluorescence.

Storage: Store the solid reagent at −20 °C in a tightly sealed vial, protected from light and atmospheric moisture. Aliquoting upon first opening is recommended to minimize repeated freeze-thaw exposure. Under recommended conditions, the product is expected to remain stable for at least 12 months, though users should verify by specific lot expiration date.

Safety: Handle as a potential irritant; wear appropriate PPE (nitrile gloves, lab coat, eye protection). Consult the product Safety Data Sheet (SDS) for full hazard classification and disposal guidance.

Reaction Protocol Overview

The following outlines a general protocol for conjugating 5-FAM hydrazide HCl to an aldehyde-bearing target. Optimize conditions empirically for each specific application.

1. Prepare the target: If labeling a glycoprotein or polysaccharide, oxidize with 1–10 mM NaIO₄ in the dark at 4 °C for 30–60 minutes, then quench with ethylene glycol and buffer-exchange to remove periodate.
2. Prepare reagent stock: Dissolve 5-FAM hydrazide HCl in DMSO or aqueous buffer to the desired concentration (e.g., 10 mM).
3. Conjugation reaction: Mix target and reagent at a molar excess of dye (typically 5–20 equivalents relative to aldehyde groups) in mildly acidic to neutral buffer (pH 5–7, e.g., 50 mM sodium acetate pH 5.5 or PBS pH 7.0–7.2). Incubate at room temperature for 1–4 hours with gentle agitation, protected from light.
4. Purification: Remove unreacted dye by size-exclusion chromatography, dialysis, or spin desalting columns appropriate for the target molecule size.
5. Characterization: Confirm labeling by measuring absorbance at ~492 nm alongside protein/nucleic acid quantification. Calculate degree of labeling (DOL) as appropriate.

Troubleshooting Common Issues

Low labeling efficiency: Confirm adequate aldehyde density on the target (increase periodate oxidation time/concentration if applicable). Ensure reaction pH is in the optimal range (pH 5–6 favors hydrazone formation kinetics). Increase dye-to-target molar ratio or extend reaction time.

High background fluorescence after purification: Incomplete removal of free dye. Repeat the purification step or use a fresh spin desalting column. Verify that the column or membrane has an appropriate molecular weight cutoff for your target.

Fluorescence signal loss over time: Hydrazone bonds are reversible under acidic aqueous conditions. Consider stabilizing with sodium cyanoborohydride (NaBH₃CN) reduction to convert the hydrazone to a stable amine. Store labeled conjugates at 4 °C protected from light.

Poor solubility: If aqueous solubility is insufficient, increase the DMSO co-solvent fraction (keep below 10% v/v in the final reaction mixture to avoid protein denaturation) or warm gently to assist dissolution.

Frequently asked questions

What type of chemistry does 5-FAM hydrazide HCl use to label biomolecules?

5-FAM hydrazide HCl reacts via its hydrazide group (–CONHNH₂) with aldehyde or ketone carbonyl groups on target molecules to form covalent hydrazone bonds. This carbonyl-selective chemistry is particularly useful for labeling periodate-oxidized glycans, oxidized lipids, and proteins bearing unnatural ketone handles.

Is 5-FAM hydrazide HCl compatible with standard FITC fluorescence filter sets?

Yes. As a fluorescein-based dye, 5-FAM hydrazide HCl has excitation and emission maxima of approximately 492 nm and 517 nm, respectively, which are well matched to standard FITC filter cubes and laser lines (e.g., 488 nm argon-ion or solid-state laser). It is therefore compatible with most fluorescence microscopes, flow cytometers, and plate readers configured for fluorescein detection.

What is the optimal pH for the hydrazide–aldehyde conjugation reaction?

Hydrazone bond formation between a hydrazide and an aldehyde is generally fastest under mildly acidic conditions, with an optimal pH range of approximately 5.0–6.0. Near-neutral pH (6.5–7.2) is also workable but may require longer reaction times or higher dye excess. Strongly alkaline conditions should be avoided, as they can reduce reaction efficiency and may affect fluorescein photophysics.

How should I store 5-FAM hydrazide HCl to maximize shelf life?

Store the solid reagent at −20 °C in a tightly sealed vial, away from light and moisture. We recommend aliquoting the material upon first opening to avoid repeated freeze-thaw cycles, which can accelerate degradation. DMSO stock solutions should also be stored at −20 °C, sealed and protected from light.

Can 5-FAM hydrazide HCl be used to detect protein carbonylation?

Yes. Carbonylated proteins, generated by oxidative stress-induced modification of amino acid side chains (particularly Pro, Arg, Lys, Thr), carry aldehyde or ketone groups that can react directly with 5-FAM hydrazide to produce a fluorescent signal. This approach offers a fluorometric alternative to the classic DNPH-based carbonylation assay, enabling gel-based or microplate-based detection of oxidative protein damage.

References

1. Zeng, Y. et al. (2009). Synthetic study of hydrazide–aldehyde bioconjugation for site-selective protein modification. General reference on hydrazone chemistry in bioconjugation. No specific DOI available from source material.
2. Hermanson, G. T. (2013). Bioconjugate Techniques, 3rd ed. Academic Press. Chapter on hydrazide chemistry and carbonyl-reactive probes.
3. Levine, R. L. et al. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. General reference; specific volume and page numbers not confirmed from source material.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.