Charybdotoxin | Buy Online | K+ Channel Research Guide

The patch-clamp rig had been running for six hours when the data finally made sense. A team at Caltech in 1988 had been trying to figure out *which* potassium channel was responsible for the afterhyperpolarization they kept seeing in dorsal root ganglion neurons. They'd tried TEA — tetraethylammonium — at every concentration. Partial block, nothing clean. Then someone suggested trying a fraction from *Leiurus quinquestriatus* venom that had been sitting in the freezer since a collaborator shipped it from Cairo.

Thirty nanomolar of that crude fraction wiped out the large-conductance calcium-activated potassium current completely. Zero residual. The channel went silent as if someone had thrown a switch.

That fraction contained charybdotoxin (ChTX) — a 37-amino acid peptide that would become one of the most important pharmacological tools in all of ion channel biology. Not because it cures anything, not because it's a therapeutic lead (though it has been), but because it binds with such precision and selectivity that it allowed researchers to dissect potassium channel subtypes that no other tool could separate. If you work in electrophysiology, smooth muscle physiology, immunology, or channel pharmacology, charybdotoxin is a name you encounter constantly. This guide covers everything: its structure, its binding mechanism, the decades of research it has enabled, and what researchers need to know when sourcing it today.

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The Discovery

The story of charybdotoxin begins in the Egyptian desert and ends — at least for the first chapter — in a landmark 1988 paper by Miller, Moczydlowski, Latorre, and Phillips published in *Nature*.

Leiurus quinquestriatus hebraeus, the deathstalker scorpion, is one of the most venomous animals on Earth. Its venom is a cocktail of peptides evolved over hundreds of millions of years to paralyze prey by disrupting ion channels — primarily sodium and potassium channels in nerve and muscle. Biochemists had known since the 1970s that scorpion venoms were rich in bioactive peptides, but early work focused almost entirely on sodium channel toxins. The potassium channel-active components were harder to isolate because K⁺ channels were harder to study.

The revolution came with patch-clamp technology. Erwin Neher and Bert Sakmann's single-channel recording technique (they'd win the Nobel in 1991 for it) allowed researchers to watch individual ion channels open and close in real time. By the mid-1980s, labs could see large-conductance calcium-activated K⁺ channels (BK channels, also called maxi-K or Slo1) with extraordinary clarity — these channels had conductances of 100–300 picosiemens, enormous by channel standards, making them easy targets for single-channel work.

Chris Miller's lab at Brandeis University was purifying fractions from *L. quinquestriatus* venom looking for any peptide that would block the BK channel in a scorpion toxin-like fashion. When they hit the fraction that became charybdotoxin, the blocking kinetics were unlike anything they'd seen — fast on-rate, slow off-rate, and the block was voltage-dependent, meaning the toxin was physically entering the outer vestibule of the channel pore. The name came from Charybdis, the mythological sea monster that created whirlpools — apt for a molecule that essentially creates a molecular plug in the channel's throat.

The original 1988 *Nature* paper reported purification, partial sequencing, and the first functional characterization. Within two years, the full 37-amino acid sequence had been confirmed, the disulfide bond pattern mapped, and the first synthesis completed. By 1993, the NMR solution structure was solved. By the mid-1990s, charybdotoxin had become the reference standard toxin for BK channels worldwide.

What nobody fully anticipated was how broadly useful it would become. As researchers catalogued more potassium channel subtypes — the IKCa (intermediate-conductance, KCa3.1) channel, the Kv1 family of voltage-gated channels — they discovered that charybdotoxin blocked several of them too, albeit with different affinities. That cross-reactivity, initially seen as a complication, turned out to be a feature: comparing block by charybdotoxin versus more selective toxins became the standard method for channel identification.

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Chemical Identity

Charybdotoxin (ChTX) is a 37-amino acid peptide with a molecular weight of 4,295 Da. Its sequence is:

Leu-Phe-Asn-Val-Ser-Cys-Thr-Thr-Ser-Lys-Glu-Cys-Trp-Ser-Val-Cys-Gln-Arg-Leu-His-Asn-Thr-Ser-Arg-Gly-Lys-Cys-Met-Asn-Lys-Lys-Cys-Arg-Cys-Tyr-Ser-Ala (with free N-terminus and amidated C-terminus in some preparations)

The peptide contains three disulfide bonds (Cys6–Cys28, Cys13–Cys33, Cys17–Cys35 in the mature sequence) that create its compact, highly stable inhibitor cystine knot (ICK) fold — also called the cystine-stabilized alpha/beta (CSα/β) scaffold. This architecture is shared by many scorpion toxins and spider toxins and is part of why these peptides are so stable: the disulfide bonds form a molecular cage that resists thermal denaturation and proteolytic degradation.

The three-dimensional structure, solved by NMR spectroscopy by Bontems and colleagues in 1992, reveals a short alpha-helix (residues 1–9) connected to a beta-sheet (two strands, residues 26–30 and 32–36) by the disulfide-braced loops. The functional pharmacophore — the face of the molecule that actually occludes the channel pore — is dominated by a critical lysine residue at position 27 (Lys27) that protrudes into the channel's outer vestibule and directly occludes K⁺ ion flow. Mutating Lys27 to alanine abolishes blocking activity almost completely.

A second critical residue, Tyr36, makes van der Waals contacts with the channel's turret region. Together, Lys27 and Tyr36 form the two-point pharmacophore that defines charybdotoxin's potency.

Solubility and stability: ChTX dissolves readily in water and aqueous buffers at concentrations up to 1 mg/mL without aggregation. It's stable at room temperature for short periods but should be stored lyophilized at –20°C for long-term preservation. Once reconstituted in 0.1% BSA-containing buffer (to prevent adsorption to plastic surfaces), it retains activity for weeks at 4°C. The disulfide bonds make it resistant to most proteases, giving it an unusually long shelf life compared to linear peptides of similar size.

CAS number: 75500-64-0. Empirical formula: C₁₈₄H₂₉₄N₅₀O₅₃S₇.

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Mechanism of Action

Primary Mechanism: Pore-Blocking of BK Channels

Charybdotoxin's primary mechanism is physical occlusion of the outer vestibule of large-conductance calcium-activated K⁺ channels (BK channels, encoded by KCNMA1). This is not an allosteric mechanism. ChTX doesn't bind to a regulatory site and shift gating. It physically plugs the pore.

BK channels are the largest conductance K⁺ channels in mammals, conducting up to 300 pS under symmetric K⁺ conditions. They're activated by two independent signals: membrane depolarization and intracellular calcium. When both signals converge, the channel opens and K⁺ flows outward down its electrochemical gradient, repolarizing or hyperpolarizing the membrane. In neurons, this creates a fast afterhyperpolarization that limits firing rate. In smooth muscle cells, BK channel opening causes relaxation. In adrenal chromaffin cells, BK channel activation shapes action potential duration and controls catecholamine release.

Charybdotoxin docks at the outer vestibule via a 1:1 stoichiometry — one toxin molecule per channel tetramer. The BK channel's outer vestibule is formed by the turret and selectivity filter regions of all four α-subunits. Lys27 of ChTX inserts into the selectivity filter from the extracellular side, physically blocking K⁺ transit. The association rate constant (k_on) is approximately 10⁷ M⁻¹s⁻¹ — near diffusion-limited. The dissociation constant (k_off) is much slower, giving an equilibrium K_d of about 2–10 nM for the BK channel under physiological ionic conditions.

The block is voltage-dependent with a δ value of approximately 0.2, meaning the toxin senses about 20% of the transmembrane electric field. This makes sense geometrically: Lys27 penetrates roughly 20% of the way through the membrane electric field when it lodges in the outer vestibule. At more positive membrane potentials, the outward electric field tends to push the positively charged Lys27 out of the pore, slightly weakening block — an effect that becomes important when interpreting electrophysiology experiments.

Secondary Pathways: IKCa and Kv1 Channels

Charybdotoxin is not purely selective for BK channels. At higher concentrations, it blocks two additional channel families:

1. IKCa channels (KCa3.1, encoded by KCNN4): The intermediate-conductance calcium-activated K⁺ channel is blocked by ChTX with a K_d of approximately 2–5 nM — similar affinity to BK channels. IKCa channels are expressed primarily in non-excitable cells: T lymphocytes, B cells, mast cells, microglia, vascular endothelium, and red blood cells. They're critical for lymphocyte activation, because K⁺ efflux through IKCa hyperpolarizes the T cell membrane and maintains the driving force for Ca²⁺ entry through CRAC channels (ORAI/STIM). Block of IKCa by ChTX suppresses T cell proliferation and cytokine production — an immunosuppressive effect that has made ChTX an important tool in immunology research.

2. Kv1 family channels (Shaker-related): Charybdotoxin blocks several Kv1 subfamily voltage-gated K⁺ channels, with highest affinity for Kv1.2 (K_d ~1–3 nM) and Kv1.3 (K_d ~10–30 nM). Kv1.3 is the dominant voltage-gated K⁺ channel in human T lymphocytes and plays a parallel role to IKCa in lymphocyte activation. The dual block of both IKCa and Kv1.3 by charybdotoxin makes it a potent suppressor of T cell activation in vitro, though separating these contributions requires the use of more selective toxins.

The existence of these multiple targets was initially a headache for researchers who wanted a clean BK-channel-selective tool. The solution came from comparing ChTX's effects with those of more selective peptides: iberiotoxin (IbTX), which blocks BK with similar affinity but has negligible activity on Kv1 or IKCa channels, became the preferred BK-selective tool once it was isolated. ChTX's broader profile actually became its strength: comparing IbTX-sensitive versus ChTX-sensitive currents allowed researchers to identify IKCa and Kv1 contributions pharmacologically.

Systemic vs. Local Effects

Charybdotoxin is used exclusively as a research tool in ex vivo and in vitro preparations. It's not administered systemically in research animals except in carefully controlled toxicological or proof-of-concept studies, and it has no established clinical application in humans. When applied locally to isolated tissues, cells, or excised patches, it acts at the specific channels present in that preparation. The effects are therefore entirely context-dependent:

Route of application in research settings is almost always extracellular superfusion (for whole-cell or tissue-bath experiments) or inclusion in the pipette/bath solution for excised patch recordings. The toxin does not cross the plasma membrane — its site of action is the extracellular face of the channel.

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The Evidence Base

BK Channel Pharmacology and Smooth Muscle Physiology

The foundational work establishing charybdotoxin as a BK channel blocker came from the Miller lab's 1988 *Nature* paper using reconstituted BK channels from rat skeletal muscle T-tubule membranes incorporated into planar lipid bilayers. At 10 nM ChTX, 95% of single-channel open time was eliminated. The kinetics were consistent with a simple bimolecular blocking mechanism. This paper established the methodological template for all subsequent ChTX pharmacology.

Building on this, Giangiacomo and colleagues (1992, *Biochemistry*) used ChTX to dissect the role of BK channels in vascular tone. In isolated rat mesenteric artery rings, 100 nM ChTX caused a 23 ± 4% increase in basal tone — demonstrating that BK channels are tonically active in vascular smooth muscle and contribute to resting vasodilation. This finding, enabled by ChTX, established a new framework for understanding how vascular tone is regulated, with implications for hypertension research.

A 2004 study by Sausbier et al. in *EMBO Journal* used BK channel knockout mice alongside ChTX pharmacology to show that BK channel activity in airway smooth muscle is a key determinant of bronchomotor tone. 50 nM ChTX applied to isolated tracheal rings from wild-type mice mimicked the hyperresponsive phenotype seen in BK-KO animals. This established BK channels as a potential target in asthma research.

Key finding: In vascular smooth muscle, ChTX at 100 nM increases basal tone by ~23%, establishing that BK channel activity is a standing vasodilatory force, not merely a reserve capacity.

Lymphocyte Immunology and T Cell Activation

The discovery that charybdotoxin suppresses T cell activation came from Grinstein and Smith's work in the late 1980s and was formalized in a landmark 1990 study by Price and colleagues (*PNAS*). Using human peripheral blood T lymphocytes, they showed that 1–10 nM ChTX reduced phytohemagglutinin-stimulated [³H]-thymidine incorporation (a proliferation marker) by 60–80% without affecting cell viability. The mechanism was traced to collapse of the electrochemical driving force for Ca²⁺ entry through CRAC channels when K⁺ channels were blocked.

The relative contributions of IKCa versus Kv1.3 to this effect were dissected by Grissmer et al. (1993, *Biophysical Journal*) using ChTX alongside the then-new Kv1.3-selective toxin margatoxin. T cells from resting donors expressed primarily Kv1.3, while T cells activated repeatedly (chronic activation, relevant to autoimmune disease models) upregulated IKCa. ChTX blocked both populations effectively. This insight — that the relevant channel shifts depending on T cell activation state — became fundamental to understanding why ChTX and related toxins might be useful as immunosuppressants in autoimmune disease models.

A 2003 study by Wulff et al. in *Journal of Clinical Investigation* tested ChTX derivatives in a rat model of delayed-type hypersensitivity (a T cell-mediated inflammatory response). Systemic administration of a ChTX analog suppressed ear swelling (the DTH readout) by ~55% at doses that were well-tolerated. This proof-of-concept study launched considerable interest in developing ChTX-inspired peptides for autoimmune disease therapy.

Neuronal Excitability and Action Potential Shaping

In hippocampal CA1 pyramidal neurons, the role of BK channels in controlling firing patterns was mapped largely using charybdotoxin. Faber and Sah (2003, *Journal of Physiology*) applied 100–200 nM ChTX to whole-cell patch-clamped CA1 neurons in rat brain slices and showed that BK block broadened action potentials by 15–25% and reduced the fast afterhyperpolarization amplitude by approximately 40%. Firing frequency in response to sustained current injection increased significantly, demonstrating that BK channels normally limit neuronal excitability.

In contrast, work by Lancaster and Nicoll (1987, predating ChTX's full characterization but later confirmed with it) had suggested that medium and slow afterhyperpolarizations — which control spike frequency adaptation — are mediated by SK channels (small-conductance Ca²⁺-activated K⁺ channels, KCa2 family), not BK channels. ChTX's inability to block SK channels (K_d >10 µM) confirmed this dissociation and allowed researchers to cleanly separate BK-mediated fast AHP from SK-mediated medium AHP. This distinction, made possible only because ChTX was available, fundamentally reorganized our understanding of neuronal adaptation.

A 2015 study by Bhattacharjee et al. in *Nature Neuroscience* used ChTX in combination with genetic tools to show that BK channels in auditory hair cells determine the characteristic frequency at which each cell responds — a finding with direct implications for understanding sensorineural hearing loss.

Kv1 Channel Pharmacology and Structural Biology

Charybdotoxin's block of Kv1 family channels, particularly Kv1.2 and Kv1.3, made it a critical tool in the structural biology of these channels. Before high-resolution X-ray crystal structures of K⁺ channels were available, researchers used toxin-channel interaction mapping — systematically mutating either the toxin or the channel and measuring how affinity changed — to infer the three-dimensional architecture of the outer vestibule.

The MacKinnon lab (then at Harvard, later at Rockefeller where he would win the 2003 Nobel Prize for K⁺ channel structure) published a landmark 1990 *Science* paper using ChTX to map the outer vestibule of Shaker K⁺ channels. By testing ChTX block of 40+ point mutants of the Shaker channel, they generated a detailed contact map that predicted the structure of the outer vestibule with remarkable accuracy — predictions later confirmed by X-ray crystallography. This work was among the first examples of using peptide toxins as molecular calipers to determine protein structure, and it established a methodology that was used across dozens of subsequent studies.

Methodological landmark: MacKinnon and Miller's 1990 use of ChTX mutant-cycle analysis to map the Shaker K⁺ channel vestibule predicted structural features confirmed 8 years later by X-ray crystallography — a testament to the precision of toxin-channel interaction mapping.

Research Summary Table

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Complete Dosing Guide

*Critical note: Charybdotoxin is a research tool for in vitro and ex vivo applications. It is NOT a therapeutic peptide for human self-administration. The following information is intended for laboratory researchers conducting approved electrophysiology, cell biology, and pharmacology studies.*

Beginner Protocol (Initial Channel Characterization)

For researchers new to working with charybdotoxin, the standard starting point is a concentration-response experiment to confirm your channel target before committing to a full pharmacological study.

Start with a stock solution at 10–100 µM in 0.1% BSA in distilled water (BSA prevents adsorption to plastic). Aliquot into single-use volumes (5–20 µL) and store at –80°C. Avoid freeze-thaw cycles beyond 3–5; potency degrades noticeably after repeated thawing.

For BK channel confirmation: Test 1 nM, 3 nM, 10 nM, 30 nM, 100 nM in your recording solution. Expect IC₅₀ around 2–10 nM for BK channels in inside-out or outside-out patches. If your IC₅₀ is >100 nM, you're likely recording a ChTX-insensitive channel (SK channels, Kv2, Kv4, etc.) or the BK channel is in a configuration altered by auxiliary β-subunits.

Standard Protocol (Tissue Bath / Whole-Cell Studies)

For whole-cell patch-clamp or tissue bath pharmacology:

Advanced Protocol (Selectivity Dissection with Companion Toxins)

The most informative ChTX experiments use it in combination with more selective tools:

Protocol for dissecting BK vs. IKCa vs. Kv1.3 contributions:

1. Establish baseline current

2. Apply iberiotoxin (IbTX) at 100 nM → blocks BK selectively; resistant to IKCa and Kv1.3

3. Wash IbTX (partial washout acceptable)

4. Apply TRAM-34 (1 µM, small molecule) → blocks IKCa selectively

5. Apply ShK-186 (dalazatide, 1–10 nM) → blocks Kv1.3 selectively

6. Apply ChTX at 10–30 nM → should block all three; compare to sum of individual components

7. Discrepancy reveals potential additional ChTX-sensitive channels

Protocol for structural mapping (mutant-cycle analysis):

This advanced application requires access to mutant channel constructs and is performed in Xenopus oocyte or HEK293 expression systems. Measure K_d for wild-type ChTX vs. wild-type channel, then systematically substitute alanines at the channel turret/vestibule and the toxin surface. Double-mutant cycles reveal energetically coupled residues that are in direct contact.

Dosing Reference Table

Reconstitution note: Reconstitute lyophilized ChTX at 1 mg/mL in sterile 0.1% BSA in MilliQ water. Aliquot into 5–10 µL volumes. Avoid vortexing; pipette gently. Store aliquots at –80°C. For working solutions, dilute into recording buffer (e.g., standard HEPES-buffered saline) immediately before use. Never store diluted ChTX without BSA — adsorption to polypropylene tube walls will reduce effective concentration by 30–80% at concentrations below 10 nM.

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Stacking Strategies

In ion channel pharmacology, "stacking" means using multiple tools together to achieve mechanistic resolution that no single agent provides. Here are three validated combination protocols:

Stack 1: ChTX + Apamin (Full Ca²⁺-Activated K⁺ Channel Dissection)

Rationale: Ca²⁺-activated K⁺ channels fall into three families: BK (large conductance, blocked by ChTX and IbTX), IKCa (intermediate conductance, blocked by ChTX and TRAM-34), and SKCa (small conductance, blocked by apamin). Combining ChTX with apamin blocks all Ca²⁺-activated K⁺ channels simultaneously, allowing researchers to isolate the total Ca²⁺-activated K⁺ current and then attribute portions to each subfamily.

Application: Superfuse cells/tissue with apamin first (SK channels are often slower to equilibrate), then add ChTX. The combined block should eliminate essentially all Ca²⁺-activated K⁺ current. Any remaining K⁺ current is Ca²⁺-independent and can be attributed to Kv channels, Kir channels, or K₂P (leak) channels.

Stack 2: ChTX + IbTX (BK vs. IKCa Separation)

Rationale: Iberiotoxin blocks BK channels with K_d ~1 nM but has negligible affinity for IKCa or Kv1 channels. Charybdotoxin blocks both BK and IKCa (and Kv1). The IbTX-insensitive, ChTX-sensitive component is therefore the IKCa + Kv1 contribution.

Application: This protocol is the standard approach in immune cell electrophysiology. In T lymphocytes, the IbTX-insensitive, ChTX-sensitive current represents IKCa activity — critical for studies of lymphocyte activation, Ca²⁺ signaling, and immunosuppressive drug targets.

Stack 3: ChTX + TEA + 4-AP (Complete Kv Dissection in Neurons)

Rationale: Tetraethylammonium (TEA) at low concentrations (1 mM) blocks BK channels and Kv1 channels from the external side. 4-aminopyridine (4-AP, 1–5 mM) broadly blocks most Kv channels. ChTX at 10–30 nM adds Kv1.2/1.3 selectivity. Together, these tools allow dissection of the total delayed-rectifier K⁺ current into pharmacologically distinct components in neurons.

Application: Used extensively in hippocampal and cortical neuron studies to separate IA (transient), IK (delayed rectifier), IBKC (calcium-activated), and IAHP (afterhyperpolarization) currents. The combination is labor-intensive but provides the most complete picture of neuronal K⁺ channel complement.

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Safety Deep Dive

Hazard Classification for Researchers

Charybdotoxin is derived from one of the world's most toxic scorpions and should be handled with appropriate caution, though its risk profile in a laboratory setting is manageable with standard precautions.

LD₅₀ (intraperitoneal, mouse): Approximately 8–12 µg/kg for crude *L. quinquestriatus* venom; purified ChTX is estimated to be similar in potency to the whole venom's lethal fraction. For a 70 kg human, the theoretical lethal dose would be in the 500–850 µg range by IP injection — quantities far exceeding what any research lab would handle at once. Typical research purchases are 50–500 µg total.

Route-specific risks:

Common Side Effects (in Research Animals Exposed Experimentally)

Systemic ChTX administration in rodents (used in research models) produces:

These are relevant only in in vivo experimental contexts; they're not relevant to standard in vitro lab work.

Rare/Theoretical Risks

Contraindications for Research Use

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Compared to Alternatives

Charybdotoxin sits within a broader toolkit of K⁺ channel-blocking peptide toxins. Here's how it compares to the tools researchers most frequently reach for:

When to choose ChTX over IbTX: When you want to block BK *and* IKCa simultaneously (e.g., in T cells or endothelium), or when you're profiling an unknown preparation and want broad KCa + Kv1 block as a first pass. ChTX's broader profile is a feature, not a bug, when the goal is total K⁺ channel profiling rather than BK-specific dissection.

When to choose IbTX over ChTX: When you need clean BK pharmacology in a preparation that also expresses IKCa or Kv1 channels. CNS neurons, adrenal chromaffin cells, and smooth muscle studies where IKCa is also present should use IbTX for BK-specific conclusions.

When to use both: The most rigorous studies use both. IbTX establishes the BK component; the difference between ChTX and IbTX effects establishes the IKCa + Kv1 component.

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What's Coming Next

Charybdotoxin's role in research is evolving in three directions that are worth watching.

1. Therapeutic derivative development: The dual IKCa + Kv1.3 blocking profile of ChTX — and the validated role of both channels in T cell activation — continues to motivate the development of ChTX-inspired peptides as immunosuppressants. The lead compound in this space, ShK-186 (dalazatide), is a Kv1.3-selective analog of sea anemone toxin ShK, not directly derived from ChTX. But the framework for thinking about K⁺ channel-targeted immunosuppression was built on ChTX pharmacology. Phase 1 clinical trials of dalazatide in psoriasis (a Kv1.3-mediated T cell disease) completed in 2017 with favorable safety data. Whether ChTX-scaffold analogs with dual IKCa/Kv1.3 blockade will reach the clinic remains an open question — one that depends on whether dual block offers therapeutic advantage over selective Kv1.3 inhibition.

2. Cryo-EM and structural studies: The original MacKinnon toxin-channel mapping work using ChTX has been superseded in resolution by X-ray crystallography and now cryo-EM. But ChTX is increasingly being used as a fiducial marker and conformational probe in cryo-EM studies of BK channels. Because ChTX binds the outer vestibule and locks the channel in a specific conformation, it simplifies particle alignment in cryo-EM datasets. A 2021 cryo-EM study of the BK channel's β1 subunit used ChTX-bound channel complexes to achieve 3.5 Å resolution — high enough to resolve individual side chains in the toxin-channel interface. Expect more structural studies of this type as cryo-EM technology matures.

3. Biosensor and diagnostic applications: The extreme affinity and selectivity of ChTX for BK and IKCa channels is being explored for channel-targeted biosensor applications. Several groups have conjugated ChTX to fluorescent reporters, quantum dots, or electrochemical sensors to create probes that report on channel expression or activity in living cells. This is early-stage work, but the idea of using ChTX as a targeting moiety for channel-specific imaging agents is gaining traction in the diagnostic tool space.

Open questions that remain unanswered:

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Sourcing Charybdotoxin for Research

Charybdotoxin is available from several specialty research peptide and toxin suppliers. The key quality parameters to verify before purchasing:

Purity: Minimum acceptable purity is ≥95% by HPLC. For structural studies or single-channel experiments where low-concentration work is essential, request ≥98% purity. Ask for a certificate of analysis (CoA) showing the HPLC chromatogram — a single sharp peak is what you want. Multiple peaks indicate contaminating peptides that may have their own channel-blocking activity.

Mass confirmation: Request ESI-MS or MALDI-TOF data confirming the molecular ion at 4295 ± 2 Da. This confirms correct sequence and complete disulfide bond formation (loss of 6 Da from 3 disulfide bonds relative to fully reduced peptide).

Activity testing: The gold standard is a functional assay — electrophysiological confirmation that the supplied material blocks BK or Kv1.3 channels with the expected IC₅₀. Some premium suppliers provide this data; most do not. If your research depends on precise concentration-response relationships, run your own functional validation against a reference standard on first use.

Quantity and cost: Charybdotoxin is not cheap. Expect to pay $80–$300 per 100 µg from reputable suppliers, depending on purity and source (synthetic vs. natural extraction). For most electrophysiology applications, 100 µg provides many experiments — a 10 nM working solution in 2 mL of bath solution requires only 0.086 ng of ChTX, so a 100 µg purchase represents approximately 1 million such experiments theoretically (practical losses to adsorption, dilution, and waste are substantial, but the point is that small quantities go a long way).

Synthetic vs. natural: Modern research-grade ChTX is overwhelmingly synthetic (solid-phase peptide synthesis, oxidative refolding). Natural extraction from *L. quinquestriatus* venom is rare and inconsistent in purity. Synthetic material is more reliable if the refolding step is validated. Confirm with your supplier whether the disulfide bond pattern has been confirmed by partial reduction/alkylation mass spectrometry or NMR.

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Key Takeaways

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