Installing a Redox Switch on Biomolecules: Principles, Conjugation Essentials, and an Application Guide for ATTO MB2 NHS ester

ATTO MB2 NHS ester can be understood as a covalently attachable “active tag” made by converting the classic redox dye methylene blue (MB) into an NHS ester that reacts with primary amines on biomolecules. Its key advantage is not “a flashier color,” but a reversible redox switch:

• In the oxidized state, it shows strong absorption at ~668 nm.
• Upon reduction, it converts to a nearly colorless leuko form (reduced/bleached state).
• Under aerobic conditions, it can be re-oxidized, and the absorption is restored.

LMB (leucomethylene blue) is continuously oxidized back to MB (methylene blue) in the presence of air/dissolved oxygen. This is why the leuko state is often difficult to maintain for long periods in air. If you need more stable ON↔OFF control, a deoxygenated environment or electrochemical potential control is typically required to drive and “lock” the redox state.

1,What is a “redox switch”?

1.1 What do oxidation and reduction mean?

• Oxidation: removing electrons (more electron-deficient).
• Reduction: adding electrons back (more electron-rich).

Methylene-blue-type dyes are a classic example: the oxidized form is blue/strongly absorbing; the reduced form becomes colorless leucomethylene blue (LMB). When exposed again to oxygen or other oxidizing conditions, it can be “recharged,” and the absorption returns. This “colored ↔ colorless” change is not simple fading—it is a reversible electron-state switch at the molecular level.

1.2 Why is it useful? Where is the real “highlight”?

Many “ordinary dyes” only tell you whether something is present and how much. Redox dyes add an extra information channel:

• Besides an optical readout (absorbance), the redox state can also be read out electrochemically—more like an “electrical circuit readout.” The methylene-blue family has long been used as a redox reporter in electrochemical biosensing.
• Electrochemical readout usually requires the probe to be immobilized near an electrode (or positioned within a controllable distance from the electrode interface). Changes in distance/conformation strongly affect electron-transfer efficiency; otherwise, signals can be weak or poorly reproducible.

2,Why “upgrade” from free methylene blue (MB) to ATTO MB2?

ATTO MB2 is a commercialized member of the “methylene blue derivative” family. Vendor notes and typical documentation generally emphasize three core advantages:

1. Retains the classic reversible redox switching of MB

• ATTO MB2 remains a typical redox dye: it can be reduced to a nearly colorless leuko form, then return to the colored/strongly absorbing oxidized state under oxidizing conditions (e.g., air/oxygen).
• Value: you gain not only an optical “present/absent or how much” signal, but a state site that can be repeatedly switched by chemical or electrochemical means.

2. Far-red absorption + high molar absorptivity

• Typical spectral constants are often given as (approximately): λabs ≈ 668 nm, εmax ≈ 1.0 × 10⁵ M⁻¹·cm⁻¹, CF260 = 0.08, CF280 = 0.24 (often reported using the carboxy reference form in aqueous solution). Here εmax is the molar extinction coefficient at the longest-wavelength absorption maximum. In practice, Amax (absorbance at that peak) is frequently used to back-calculate dye concentration and to compute DOL (degree of labeling).
• Note: the exact λabs/ε can shift slightly with solvent, buffer system, and pH (often on the few-nanometer scale).

3. More “usable” properties: controllable solubility/charge + covalent installability

• ATTO MB2 is moderately hydrophilic and cationic; after coupling to a substrate, the dye moiety is often described as having a net charge of about +1.
• Important: +1 here refers to the dye moiety. The overall charge and nonspecific interactions of the final conjugate still depend strongly on the substrate (protein/nucleic acid) surface properties and charge.
• ATTO MB2 provides a reactive chemical handle (e.g., NHS ester) that enables a stable, reproducible covalent installation of the redox tag onto the target molecule—reducing drift/leaching/background from free dye and enabling more standardized experimental design and controls.

Summary: an upgrade from “free MB” to a covalently installable, more standardized redox-switch label (the ATTO MB2 family).

3,What is an NHS ester? The chemical “interface” that lets the dye be “welded onto” molecules

If ATTO MB2 is a “chip with a switch,” then the NHS ester (N-hydroxysuccinimide ester) is its “solder pad”—a widely used chemical interface for covalently attaching dyes to biomolecules.

3.1 What does it mainly react with?

The most important and common use of NHS esters is reaction with primary amines (–NH₂) on biomolecules to form a stable amide bond. In proteins, primary amines most commonly come from:

(a) the N-terminus

(b) lysine side chains (ε-amino groups)

(c) Note: other primary-amine-containing substrates—such as amino-modified nucleic acids, aminated polysaccharides, or small molecules—are also applicable.

3.2 Why is pH ~8 emphasized, and why avoid Tris?

(1) pH 8.0–8.5: make amines more reactive, but don’t go too alkaline

• Under mildly basic conditions (pH ~8.0–8.5), amines are more likely to be in a reactive (nucleophilic) state, improving coupling efficiency.
• Reminder: higher pH also accelerates hydrolysis of NHS esters in water. So “more basic” is not always better. A common approach is mild basic buffering, controlled reaction time, and minimizing the hydrolysis window.

(2) Avoid Tris/glycine/ethanolamine and other “free-amine” systems: the buffer will compete

• Tris, glycine, ethanolamine, etc. contain free primary amines and will compete with your substrate for the NHS ester—effectively consuming dye in the buffer and lowering labeling efficiency.
• Avoid buffers/additives containing free amines. If your sample is in such a system, typically perform desalting/dialysis/gel filtration into an amine-free buffer before coupling.
• Some ammonia/ammonium systems can also introduce competition or interference under certain conditions; experimentally, bicarbonate/carbonate buffers are often preferred for NHS coupling.
• Note: after the reaction, if you need to quench/cap remaining NHS ester, you can intentionally add Tris or ethanolamine as a quenching reagent—the key is that they should appear after coupling, not during coupling.

(3) Can PBS/phosphate buffer be used?

• PBS/phosphate can be used for sample storage or pre-treatment (e.g., dialysis/desalting). However, for the NHS coupling step, it is common to switch into bicarbonate/carbonate (or borate) buffer and adjust to about pH 8.3–8.5 to obtain more consistent coupling efficiency and a more controllable hydrolysis window.

4,Which biomolecules can be “switched on”?

ATTO MB2 NHS ester can be used to label DNA, RNA, and proteins (including antibodies).

Note: because it is an amine-reactive NHS ester, for nucleic acids this usually means primary-amine-modified DNA/RNA (especially oligonucleotides). Otherwise, you must first introduce a reactive amine handle before coupling.

1. For protein/antibody labeling, the key is “couple to available amines,” but the number and accessibility of reactive amines vary by protein. For first-time labeling, it is recommended to test a small gradient of dye:protein molar ratios.

Typical conditions: 0.1 M bicarbonate buffer, pH preferably 8.3, room temperature for 30–60 min. A common practical note is: do not dilute the protein too much—~2 mg/mL is recommended; below 2 mg/mL labeling efficiency may decrease.

2. Oligonucleotides are commonly labeled by reacting an amino-modified oligo with an NHS ester, typically in carbonate buffer pH 8–9 for about 2 h at room temperature. If longer reaction time is needed, lowering pH to 7–7.5 is recommended to reduce hydrolysis/side reactions.

Whether labeling proteins/antibodies or amino-modified oligos, after the reaction you should remove unreacted (free) dye and hydrolyzed dye as soon as possible. Otherwise, background rises and concentration/DOL calculations are distorted by “free dye.”

• For proteins/antibodies, gel filtration/desalting columns (e.g., Sephadex G-25) are commonly used to separate labeled biomolecules from free dye.
• For oligonucleotides, gel filtration or reversed-phase HPLC can provide higher purity.

After purification, use the manual’s correction factors (CF260/CF280) to subtract the dye’s absorbance contribution at 260/280 nm from A260/A280 (e.g., A260_corr = A260 − CF260×Amax; A280_corr = A280 − CF280×Amax, where Amax is the absorbance at the dye’s absorption maximum), then use the corrected concentration to compute DOL more reliably.

5,ATTO MB2 NHS ester: experimental checklist

Stage	What you do	Recommended conditions/parameters	Most common problem	Quick self-check
Before reaction | Buffer system	Switch to an amine-free coupling buffer	Protein: 0.1 M bicarbonate, pH preferably 8.3; solution must not contain Tris/glycine/ammonium salts	Using Tris/glycine → dye gets “consumed” by the buffer	No free amines (Tris, amino acids, etc.) in the reaction
Before reaction | Dye dissolution	Prepare dye stock fresh; minimize water exposure	Amine-free, dry DMF or DMSO; prepare fresh to reduce hydrolysis	DMSO/DMF absorbs moisture → NHS hydrolysis/inactivation; some guidance notes DMF may generate amine impurities upon storage, consuming active ester	Stock is clear, freshly prepared, kept as dry as possible
Before reaction | Substrate concentration	Don’t dilute protein too much	Protein recommended 2 mg/mL; <2 mg/mL reduces labeling efficiency	Protein too dilute → fewer effective collisions; hydrolysis dominates	Confirm protein concentration ≥2 mg/mL (or as close as possible)
During reaction | Time & ratio	Start with a small optimization range	Suggested: RT 30–60 min; first run: test different dye:protein ratios; DOL 1–2 as a starting target	Chasing high DOL immediately → loss of activity/aggregation/high background	Run 2–3 small ratio gradients first
After reaction | Remove free dye	Thoroughly remove “free dye”	Gel filtration/SEC (recommended Sephadex G-25); typically labeled protein elutes first, free dye later	Incomplete purification → high background; wrong quantitation	Check elution: are there “two colored/absorbance peaks,” and are fractions collected correctly?
Reading & correction | CF260/CF280	Correct “dye contribution” in A260/A280	ATTO MB2: CF260=0.08, CF280=0.24	Mistaking dye absorbance at 260/280 for nucleic acid/protein	Quick formula (same dilution): (1) A260,corr = A260 − CF260 × Aλmax (2) A280,corr = A280 − CF280 × Aλmax where Aλmax is absorbance at the dye’s λmax (ATTO MB2 ~668 nm)
Storage | Reagent & conjugate	Store per manual/protein practice	Reagent: protect from light/moisture, −20°C; conjugate: follow protein storage norms, aliquot and freeze for long-term storage to avoid freeze–thaw; centrifuge briefly before use to remove aggregates	Freeze–thaw/aggregation → poorer activity and reproducibility	Aliquot, protect from light; quick spin before use

Note on protein concentration: Prefer following the manual recommendation and prepare protein at ~2 mg/mL if possible. If the sample is truly more dilute, you may compensate by increasing dye molar ratio, reducing volume, and shortening the hydrolysis window—but do small gradients to validate.

Key parameter comparison: protein/antibody vs amino-oligo

Item	Protein/antibody	Amino-modified oligonucleotide (amino-oligo)
Buffer	0.1 M bicarbonate, pH preferably 8.3	0.2 M carbonate buffer, pH 8–9
Substrate concentration note	Recommended ≈ 2 mg/mL	Example: 0.1 mM amino-oligo (illustrative)
Dye solvent	Dry, amine-free DMF/DMSO; prepare fresh	Dry DMF; example 5 mg/mL
Reaction time	RT 30–60 min	RT ~2 h; if longer, lower pH to 7–7.5
Purification	Gel filtration (recommended Sephadex G-25)	Gel filtration or reversed-phase HPLC

6,Two levels of application highlights for the ATTO MB2 “switch label”

Application level	What it enables	“Differentiation” provided by the label	Typical uses
Level 1: Traceability + redox-switch controls	Turn the target into a “switchable absorbance tag”	Far-red absorbance + reversible redox switching: reduction → near-colorless leuko; oxidation → absorbance restored (ON↔OFF)	Method validation/controls/troubleshooting: confirm successful coupling, verify purification cleanliness, confirm signal changes arise from the labeled molecule rather than background; also usable for tracing and workflow testing
Level 2: Electrochemical readout (advanced)	One tag, readable by both optics and electricity	As a redox reporter: immobilized near an electrode, SWV (etc.) can read current; conformational/distance changes → altered electron-transfer efficiency → current changes	Electrochemical biosensing: attach ATTO MB2 to the end of DNA/aptamer probes as an electrochemical output, enabling electrical detection of hybridization or target binding

Summary: The same “switch label” can be read optically (absorbance) and—on the right platform—electrochemically. This dual-mode capability is its core value compared with ordinary dyes.

7,Navigation Table | ATTO MB2–Related Products: Choose the “Use Path” First, Then Locate Items in the Corresponding Product Table

Typical need / scenario	Which table to check first	Why this table fits best	Key “category keywords” to focus on in the table
Need a redox-responsive dye: want to write “reversible redox / methylene-blue derivative” features into the protocol; build a redox-responsive / recoverable labeling or probe system	Table 1	The key product Atto MB2 NHS ester is in Table 1; redox systems are often selected together with “covalent labeling / bioorthogonal / intermediates” for a complete workflow	Redox active ester (MB2); Amine labeling | NHS ester
Routine amine labeling for proteins/antibodies: random labeling of Lys/N-terminus primary amines; fluorescent antibodies, protein tracing, standard multicolor panels	Table 1	Table 1 consolidates NHS ester series across wavelengths—the most common entry point for primary-amine coupling	Amine labeling | NHS ester (active ester) (390/425/465/488/…/740)
More “site-specific” protein labeling: mainly label Cys sites to reduce randomness and improve site control (e.g., site-specific conjugation, more uniform probes)	Table 1	Table 1 includes two thiol routes: Maleimide and Iodoacetamide. The former forms stable thioether bonds via addition; the latter alkylates/caps or labels thiols	Thiol labeling | Maleimide (site-specific); Thiol labeling | Iodoacetamide (alkylation)
Click chemistry / bioorthogonal labeling: target already has azide/alkyne, or need fast copper-free reactions (live cells, rapid labeling, chemical biology)	Table 1	Both Click (Azide/Alkyne) and IEDDA (Tetrazine) are in Table 1—an orthogonal “install a handle first, then add the fluorophore” strategy	Click | Azide / Click | Alkyne; Bioorthogonal | Tetrazine (IEDDA)
Glycan / carbonyl-site labeling: oxidize glycans to generate aldehydes/ketones, then attach dye (glycoprotein/polysaccharide labeling)	Table 1	Hydrazide is designed for carbonyl (aldehyde/ketone) coupling; a classic route is labeling after glycan oxidation	Carbonyl/Glycan | Hydrazide
Don’t want ready-made NHS/Mal/Click: already have dye intermediates or want custom linkers/reaction routes (DIY conjugation, derivatization)	Table 1	Table 1 includes “re-couplable/intermediate” categories: acids, carboxyl-terminated dyes, amine-terminated dyes, spacer amines—useful for secondary activation or linker optimization	Intermediates / re-couplable | free acid/carboxylic acid; carboxyl-terminated; spacer amine; amine-terminated derivatives
Have a biotinylated target and want detection/enrichment/signal amplification: use the biotin–streptavidin system as a universal detection format	Table 2	Table 2 is the “affinity system section”: includes both biotin–dye conjugates and streptavidin–dye ready-to-use probes for rapid setup	Affinity system | Biotin; Affinity system | Streptavidin ready-made probes
Need secondary probes / universal detection reagents: don’t want to conjugate streptavidin and dye yourself; want ready-to-use reagents	Table 2	Streptavidin ready-made probes in Table 2 can recognize any biotinylated molecule directly, eliminating conjugation steps and offering maximum universality	Streptavidin ready-made probes (488/647)
Want “ready-to-use” finished probes: no chemical conjugation, just fast staining/tracing/marking of specific targets	Table 3	Table 3 is entirely “finished application probes”: lipid membrane probes, dextran tracers, F-actin staining, etc.—ideal for quick experiments/controls	Finished probes | membrane labeling (lipid); Finished probes | tracing (dextran); Finished probes | cytoskeleton (F-actin)
Method development / instrument / spectral controls: need parent dyes for brightness, spectra, stability, system calibration or controls	Table 3	Table 3 includes “parent dye / control” items—good for basic optical controls or as references for later derivatization	Parent dyes / controls
Label EVs / liposomes / vesicles / membrane systems: want dyes that associate with lipids as “ready-made membrane probes” for direct use	Table 3	Atto 647N DOPE is a finished lipid probe commonly used for direct membrane/vesicle/EV labeling without additional conjugation	Finished probes | membrane labeling (lipid)

Summary:

• For chemical conjugation / probe construction → start with Table 1 (NHS / Maleimide / Click / IEDDA / Hydrazide / intermediates).
• For biotin–streptavidin universal detection systems → see Table 2 (Biotin and Streptavidin ready-made probes).
• For ready-to-use finished probes / methodological controls → see Table 3 (DOPE lipid, dextran tracing, F-actin staining, parent dye controls).

Table 1 | Redox Active Ester (MB2) + Covalent Labeling/Bioorthogonal + Intermediates (Re-couplable)

Category	Aladdin Cat. No.	Name	CAS No.	Spec / Purity	Product features & applications
Redox active ester (MB2)	A755437	Atto MB2 NHS ester	——	≥90% (HPLC), Methylene Blue derivative	Primary-amine labeling (NHS ester); reversible redox methylene-blue derivative dye, suitable for redox-responsive/recoverable labeling and probe systems
Amine labeling | NHS ester (active ester)	A1454184	ATTO 390 NHS ester	914203-48-8	——	Protein/antibody Lys/N-terminal primary-amine labeling; short-wavelength channel control / multicolor pairing
Amine labeling | NHS ester (active ester)	A334800	Atto 425-NHS ester	892156-28-4	≥90%	Mainstay for primary-amine labeling; suitable for early-channel use/controls in multicolor panels
Amine labeling | NHS ester (active ester)	A1452633	ATTO 465 NHS ester	1173020-88-6	——	Primary-amine labeling; commonly used in blue–green transition channels for multiplex staining
Amine labeling | NHS ester (active ester)	A1454649	ATTO 488 NHS ester	863772-22-9	——	Mainstay for primary-amine labeling; classic 488 channel choice, widely used in microscopy/flow cytometry
Amine labeling | NHS ester (active ester)	A1451493	ATTO 514 NHS ester	——	——	Primary-amine labeling; green/yellow-green channel option for multicolor separation
Amine labeling | NHS ester (active ester)	A1453944	ATTO 532 NHS ester	924660-19-5	——	Primary-amine labeling; green–yellow region, helps separate spectra from 488/594
Amine labeling | NHS ester (active ester)	A1452582	ATTO 550 NHS ester	1005770-00-2	——	Mainstay for primary-amine labeling; commonly used orange-red channel, helpful for lower background/multicolor panels
Amine labeling | NHS ester (active ester)	A1451834	ATTO 594 NHS ester	——	——	Primary-amine labeling; common red channel, suitable for 488/647 combinations
Amine labeling | NHS ester (active ester)	A1454083	ATTO 610 NHS-ester	——	——	Primary-amine labeling; extended red region channel for panel expansion
Amine labeling | NHS ester (active ester)	A1451326	ATTO 620 NHS-ester	——	——	Primary-amine labeling; red region channel option for panel expansion
Amine labeling | NHS ester (active ester)	A1452179	ATTO 633 NHS ester	2982226-58-2	——	Mainstay for primary-amine labeling; red/far-red entry channel, common in imaging/flow
Amine labeling | NHS ester (active ester)	A1450977	ATTO 647 NHS ester	——	——	Mainstay for primary-amine labeling; far-red with low background, suitable for tissues/thick samples
Amine labeling | NHS ester (active ester)	A1452008	ATTO 665 NHS ester	——	——	Primary-amine labeling; longer-wavelength far-red channel, helps reduce autofluorescence interference
Amine labeling | NHS ester (active ester)	A1451482	ATTO 680 NHS ester	1537182-26-5	——	Primary-amine labeling; near-IR direction choice, suited for low-background/deeper-imaging trends
Amine labeling | NHS ester (active ester)	A1452925	ATTO 700 NHS ester	——	——	Primary-amine labeling; near-IR channel, helps reduce scattering and background (instrument-dependent)
Amine labeling | NHS ester (active ester)	A1451806	ATTO 725 NHS-Ester	——	——	Primary-amine labeling; longer near-IR channel for multicolor strategies in high-background samples
Amine labeling | NHS ester (active ester)	A1453577	ATTO 740 NHS ester	1448146-89-1	——	Primary-amine labeling; longer near-IR wavelength for extending far-red/near-IR panels
Thiol labeling | Maleimide (site-specific)	A1454075	ATTO 488 maleimide	1219127-42-0	——	Selective addition to Cys thiols forming stable thioether bonds; for more site-specific protein labeling
Thiol labeling | Maleimide (site-specific)	A1452568	ATTO 550 maleimide	870534-97-7	——	Site-specific thiol labeling; orange-red channel “site tag” for multiplex workflows
Thiol labeling | Maleimide (site-specific)	A1452642	ATTO 647 maleimid	——	——	Site-specific thiol labeling; far-red channel for low-background site-specific conjugation
Thiol labeling | Maleimide (site-specific)	A1453943	ATTO 680 maleimide	2413537-75-2	——	Site-specific thiol labeling; near-IR channel for low-background/deeper-imaging trends
Thiol labeling | Iodoacetamide (alkylation)	A1451526	ATTO 488 iodacetamid	2305285-47-4	——	Thiol alkylation (Cys) route; suitable for systems requiring strong covalent capping/labeling of thiols
Click | Azide (click)	A1451317	ATTO 550 azide	——	——	Click chemistry to attach dye onto alkyne/cyclooctyne-bearing substrates; for bioorthogonal labeling schemes
Click | Alkyne (click)	A1452030	ATTO 550 alkyne	——	——	Click pairing with azide substrates; suitable for chemical biology / controlled conjugation systems
Click | Azide (click)	A1454096	ATTO 700 Azide	——	——	Near-IR click chemistry version; suitable for low-background click labeling
Click | Alkyne (click)	A1452924	ATTO 700 Alkin	——	——	Near-IR click chemistry version; used paired with azide
Bioorthogonal | Tetrazine (IEDDA)	A664762	ATTO 565 (tetrazine (MeTet)	——	——	Fast copper-free IEDDA bioorthogonal reaction (often paired with TCO, etc.); suitable for live-cell/rapid labeling strategies
Carbonyl/Glycan | Hydrazide	A1451230	ATTO 488 hydrazid	——	——	Reacts with aldehydes/ketones (often for carbonyl sites after glycan oxidation); common approach for glycoprotein/polysaccharide labeling
Intermediates / re-couplable | free acid / carboxylic acid	A1505315	ATTO 425 Acid	652966-03-5	——	Free-acid dye; can be used as a control or for custom conjugation/derivatization routes
Intermediates / re-couplable | carboxyl-terminated	A1454646	ATTO 488 carboxylic acid	1443553-08-9	——	Carboxyl-terminated derivative; can be further activated (e.g., EDC/NHS) for coupling to amines
Intermediates / re-couplable | spacer amine	A1452679	ATTO 550 cadaverin	——	——	Spacer-bearing amine derivative; reduces steric hindrance and improves accessibility for downstream coupling
Intermediates / re-couplable | amine-terminated derivative	A1452219	ATTO 665 amine	——	——	Amine-terminated intermediate; for custom synthesis/conjugation or specific chemical-biology linker strategies

Table 2 | Affinity System (Biotin / Streptavidin)

Category	Aladdin Cat. No.	Name	CAS No.	Spec / Purity	Product features & applications
Affinity system | Biotin	A1454599	ATTO 488 biotin	——	≥98%	Biotinylated probe; binds (strept)avidin with high affinity for detection/enrichment/signal amplification
Affinity system | Biotin	A478508	Atto 647N- Biotin	——	BioReagent, suitable for fluorescence analysis, ≥90% (HPLC)	Far-red biotinylated probe; suitable for low-background biotin–streptavidin detection systems
Affinity system | Streptavidin ready-made probe	A1453279	ATTO 488 streptavidin	1430816-27-5	——	Ready-made streptavidin–dye secondary probe; directly recognizes biotinylated targets
Affinity system | Streptavidin ready-made probe	A1454097	ATTO 647 streptavidin	——	——	Far-red streptavidin ready-made probe; low-background detection/imaging for biotinylated systems

Table 3 | Finished Probes + Parent Dyes / Controls

Category	Aladdin Cat. No.	Name	CAS No.	Spec / Purity	Product features & applications
Finished probe | Membrane labeling (lipid)	A755469	Atto 647N DOPE	——	Suitable for fluorescence analysis	Ready-to-use lipid probe for labeling membranes/liposomes/vesicles/EVs; no additional conjugation required—ready to load/use directly
Finished probe | Tracing (dextran)	A290987	ATTO488™-Dextran	——	4 kDa	Low-molecular-weight dextran finished probe; commonly used for endocytosis/diffusion/tracing and control experiments
Finished probe | Tracing (dextran)	A291034	ATTO647n™-Lysine-Dextran	——	70 kDa	High-molecular-weight far-red dextran finished probe; used for endocytosis/tracing/bulk-phase distribution studies
Finished probe | Cytoskeleton (F-actin)	P755454	Phalloidin–Atto 647N	——	BioReagent, suitable for fluorescence analysis, ≥80% (HPLC)	Ready-to-use F-actin staining probe; far-red channel, suitable for multicolor co-staining with 488/550, etc.
Parent dye / control	A386778	Atto 590	——	≥95%, mixture of isomers	Parent dye/control (mixture of isomers); can be used for spectral/staining controls or further derivatization
Parent dye / control	A664743	ATTO Rho6G	——	——	Rhodamine-family parent dye; commonly used for fluorescence performance, instrument, and methodological controls (depending on experimental goals)

8,Overview of Supporting Chemicals for ATTO MB2 NHS ester Experiments: Buffers/Solvents/Chromatography × Coupling & Quenching × Redox Controls & Validation

Mini-Table 1 | Buffer salts / basic medium components + quenchers / stabilizers (commonly used for coupling, washing, and storage)

Category	CAS No.	Aladdin Cat. No.	Name	Spec / Purity	Product features & role
Buffer salts / basic medium components (ionic strength)	7647-14-5	C111542	Sodium chloride	For plant cell culture, ≥99.5%	Used to prepare PBS/saline and wash solutions; used for post-coupling washing, stabilizing ionic strength, and reducing nonspecific adsorption.
Buffer salts / basic medium components	7447-40-7	P112144	Potassium chloride	For cell culture, ≥99.5%	Used to prepare PBS/cell buffers and maintain ionic strength; used for post-coupling washing and system stabilization.
Buffer salts / basic medium components	12125-02-9	A116373	Ammonium chloride	For cell culture	Common inorganic salt for media/buffer formulations and ionic strength control; often used for system setup and controls (not an essential coupling reagent). Note: NH₄Cl can be used in culture/other steps, but during NHS coupling the manual typically recommends avoiding ammonium-salt systems.
Buffer salts (phosphate system)	7778-77-0	P113042	Potassium dihydrogen phosphate	For plant cell culture, ≥99%	Used with K₂HPO₄/Na₂HPO₄, etc. to prepare phosphate buffers; for coupling/washing/storage (avoid primary-amine–containing buffers).
Buffer salts (phosphate system)	7758-11-4	D433945	Dipotassium hydrogen phosphate	Anhydrous, AR, suitable for analysis	Used with KH₂PO₄ to prepare phosphate buffers; for coupling/washing/storage.
Buffer salts (phosphate system)	7558-80-7	S108343	Sodium dihydrogen phosphate, anhydrous	For cell culture/insect cell culture, ≥99% (T)	Used to prepare phosphate buffers; for pre-/post-reaction sample handling, washing, and storage.
Buffer salts (phosphate system)	7558-79-4	S118441	Disodium hydrogen phosphate, anhydrous	For cell culture/insect cell culture	Used with NaH₂PO₄ to adjust pH and form phosphate buffer systems; for post-coupling washing/storage.
Coupling buffer system (bicarbonate)	144-55-8	S118660	Sodium bicarbonate	For cell culture/insect cell culture, ≥99.5%	Classic buffer salt for NHS ester amine labeling: NaHCO₃ (pH ≈ 8.3) provides a mildly basic environment to enhance amine nucleophilicity while balancing hydrolysis.
Coupling buffer system (carbonate)	497-19-8	S432764	Sodium carbonate	Anhydrous, AR, suitable for analysis	Used to prepare carbonate buffers (more alkaline pH); can provide basic conditions for NHS coupling (higher pH increases hydrolysis—balance efficiency vs. hydrolysis).
Buffer system (boric acid/borate)	10043-35-3	B111605	Boric acid	For cell culture/insect cell culture, ≥99.5%	Used to prepare borate buffers (often at mildly basic pH); can be used for some labeling/washing conditions and methodological controls.
Buffer system (borate)	1303-96-4	S112464	Sodium tetraborate decahydrate	Chemical pure (CP), ≥99%	Common salt for borate buffer systems; used for reactions/washes/controls under mildly basic pH.
Quenching/termination (primary-amine capping agent)	141-43-5	E103809	Ethanolamine	For cell culture, ≥99%	Primary-amine quencher/capping reagent: used after coupling to cap residual NHS ester/activated sites and reduce subsequent nonspecific reactions.
Termination/capping (small-molecule primary amine)	56-40-6	G432934	Glycine	UltraBio™, molecular biology grade, ultrapure, ≥99% (NT)	Contains a primary amine: can quench/cap residual active esters; also commonly used in buffers/electrophoresis systems (avoid as a main buffer during the coupling reaction itself).
Stabilizer/solubilizer (sample storage)	56-81-5	G116209	Glycerol	For cell culture/insect cell culture, ≥99% (GC)	Storage stabilizer for proteins/probes (cryoprotection, reduced aggregation); used in storage/loading buffer formulations for labeled proteins/probes.

Mini-Table 2 | Solvents + chromatography/MS additives + reaction termination/condition adjustment (LC-MS/HPLC and post-processing)

Category	CAS No.	Aladdin Cat. No.	Name	Spec / Purity	Product features & role
Solvent / chromatography–MS (mobile phase)	75-05-8	A433526	Acetonitrile solution	MS grade, UltraPureChrom™, UHPLC grade, contains 0.1% (v/v) formic acid	Common organic phase for LC-MS/UPLC; used for analysis and purification method development for free dye and conjugates (0.1% FA supports ESI ionization).
Solvent (dye dissolution/stock solutions)	67-68-5	D103280	Dimethyl sulfoxide (DMSO)	Pharmaceutical grade, PharmPure™	Common polar solvent: used to prepare high-concentration ATTO MB2 NHS ester stock solutions and add small amounts into reaction mixtures (reduced hydrolysis, improved solubility).
Solvent (anhydrous coupling system)	68-12-2	D119450	N,N-Dimethylformamide (DMF)	Anhydrous, ≥99.8%	Anhydrous polar solvent: used to dissolve NHS-ester dyes and for low-water-activity coupling/derivatization and method development (reduces hydrolysis).
Acid / mobile-phase additive (termination/purification)	76-05-1	T103293	Trifluoroacetic acid (TFA)	For protein sequencing, ≥99%	Common acidic additive for peptide/protein HPLC; can also rapidly terminate amine coupling by acidification (protonates amines and suppresses reaction).
Acid / mobile-phase additive (pH adjustment)	64-19-7	A116166	Glacial acetic acid	AR, ≥99.5%	Used to prepare acetate systems and fine-tune pH; can also terminate reactions/post-process samples (protonates amines, suppressing further NHS-ester reactions).
Base / catalyst (non-nucleophilic base)	121-44-8	T431607	Triethylamine	For protein sequencing, ≥99.5% (GC), ampoule	Tertiary amine base: used for pH fine-tuning and certain coupling/chromatography conditions; does not provide a primary amine site that is directly acylated by NHS esters (but can still affect hydrolysis rate via pH).
Chromatography buffer / ion-pairing (oligo HPLC)	5204-74-0	T755551	Triethylammonium acetate, 1 M solution	Ready-to-use buffer solution used for the purification of chemically synthesized oligonucleotides by HPLC. Has a pH of 7.0.	TEAA ion-pair system: used for HPLC purification of oligonucleotides/nucleic-acid samples (including dye-labeled species), especially in reversed-phase/ion-pair methods.

Mini-Table 3 | Coupling reagents + redox/control systems + model materials/quality standards (system setup and validation)

Category	CAS No.	Aladdin Cat. No.	Name	Spec / Purity	Product features & role
Coupling reagent (carboxyl–amine coupling)	25952-53-8	A638729	Aladdin™ EDC	Analytical reference standard	Carbodiimide for activating carboxyl groups for coupling to amines (often used with NHS/water-soluble NHS); can be used to build control routes with “carboxyl-terminated dyes/carriers,” or for derivatization/secondary coupling.
Coupling additive (active ester formation)	6066-82-6	H109330	N-Hydroxysuccinimide (NHS)	≥98%	Used with EDC to convert activated carboxyl groups to NHS esters to improve amine-coupling efficiency; also used to build control routes/secondary coupling beyond the MB2 NHS-ester route.
Coupling additive (water-soluble active ester)	106627-54-7	H109337	Sulfo-N-hydroxysuccinimide sodium salt	≥98%	Water-soluble NHS-type additive: improves efficiency and reduces side reactions in aqueous EDC coupling; suitable for constructing control conjugation systems under more aqueous conditions.
Carrier / model substrate (polysaccharide)	9004-54-0	D490149	Dextran	Reagent grade	Model macromolecule/carrier: can serve in control routes such as “oxidize to aldehydes → couple via hydrazide/amine”; also used as a reference material for tracing systems.
Polymer / substrate (system construction)	9003-05-8	P754403	Polyacrylamide (PAM)	Anionic; MW: 16000–18000 kD; hydrolysis degree: 30–40%	Polymeric matrix/additive: used to build high-viscosity systems, polymer-environment controls, or condition screening for interactions with amine/charged systems (for system design/controls).
Reducing agent (redox switching/control)	7775-14-6	S434000	Sodium dithionite	Suitable for analysis, reagent grade	Strong reducing agent: reduces methylene-blue/MB2 systems to the colorless leuko form; used for reversible redox switching experiments and absorbance/signal-recovery controls.
Reducing/antioxidant (redox control)	50-81-7	L432790	L-Ascorbic acid	UltraBio™, ultrapure, ≥99.5% (RT)	Mild reductant/antioxidant: supports generating reduced states in MB2/MB systems and provides antioxidative protection; can reduce signal drift caused by oxidative stress.
Dye control (parent/reference)	61-73-4	M134389	Methylene blue	≥70%	Parent-family dye control for ATTO MB2: provides baseline reference for absorbance, redox reversibility, spectra, and redox behavior.
Dye control (homolog/batch control)	122965-43-9	M190219	Methylene blue (lan)	≥96%	Alternative/control source related to methylene-blue dyes: used for spectral and redox behavior comparison with MB2/MB (specific identity/properties should follow the product documentation).
Redox control (ferricyanide/ferrocyanide system)	14459-95-1	P432115	Potassium hexacyanoferrate(II) trihydrate	Ph.Eur, suitable for analysis, ACS, reagent grade	[Fe(CN)₆]⁴⁻: paired with hexacyanoferrate(III) to form a classic reversible redox couple; used for redox cycling and electron-transfer controls for MB2/MB systems. Note: avoid handling ferro/ferricyanides under strong acid; do not mix directly with TFA or concentrated acids—keep neutral to mildly basic and dispose per lab safety rules.
Redox control (ferricyanide/ferrocyanide system)	13746-66-2	P485844	Potassium hexacyanoferrate(III)	Ph.Eur, suitable for analysis, ACS, reagent grade	[Fe(CN)₆]³⁻: mild oxidant; used to re-oxidize reduced dyes back to the colored state for redox response/recovery curve controls. Same note as above.
Redox / nucleic-acid system control (Ru(III) complex)	14282-91-8	H302860	Hexaammine ruthenium(III) chloride	≥98%	A typical reversible redox complex and one of the commonly used electrochemical probes in nucleic-acid systems; can be used as a reference reagent for redox controls and electron-transfer/electrochemical readout conditions (scheme-dependent).
Analytical / QC standard	84-65-1	A119546	Anthraquinone	Melting point standard	Melting point standard for QC and instrument/method verification (supports laboratory standardization of analytical workflows).

Note: The above are representative Aladdin products. For more specifications, please refer to the full product list at the end of the document or search the Aladdin website by product name/CAS number.

Aladdin: https://www.aladdinsci.com/