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| Targets |
Connexin 43 (Cx43) hemichannels and gap junctions. Gap 26 is a connexin mimetic peptide that targets the first extracellular loop (E1 domain) of the gap junction protein connexin 43. By binding to this region, Gap 26 inhibits the opening of Cx43 hemichannels and prevents the formation of functional gap junctions, thereby blocking direct intercellular communication. Gap 26 does not affect the expression level or trafficking of connexin 43 to the plasma membrane; rather, it acts as a selective blocker of channel activity. The peptide contains the SHVR amino acid motif, which is essential for its binding and inhibitory action. Inhibition of Cx43 gap junctions disrupts electrical coupling, metabolic coupling, and signaling molecule exchange (including small molecules such as Ca2+, IP3, cAMP, ATP, and glucose) between adjacent cells, allowing researchers to dissect the functional roles of GJIC.
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| ln Vitro |
In the three cell lines (SV-ARBEC, ECV304, and RBE4), gap 26 (0.25 mg/mL, 30 min) lowers the wave size. The InsP3-triggered ATP response is totally eliminated by Gap 26 (0.25) mg/mL, 30 min, and the ATP release is lowered to below the control level, suggesting that the basal ATP release is also impacted[1]. Although connexin 43 was present in the cell lines employed, gap 26 did not affect dye coupling through junctional channels, as demonstrated by the FRAP experiments[1]. However, gap 26 does severely decrease our InsP3-triggered intercellular calcium waves. The rabbit superior mesenteric arteries' rhythmic responses are dose-dependently reduced by gap 26 (100-300 μM), with an IC50 of 28.4 ± 3.4 μM[2].
Gap 26 TFA is a connexin mimetic peptide that blocks gap junctions and hemichannels composed of connexin 43. In vitro, Gap 26 inhibits dye transfer between adjacent cells in gap junction coupling assays. In scrape loading/dye transfer assays, cells (e.g., NRK-49F, HeLa cells expressing Cx43, cardiac myocytes, or astrocytes) are grown to confluence, a scratch is made with a scalpel blade, and the cells are incubated with a fluorescent gap junction-permeable dye (e.g., Lucifer Yellow CH (457 Da) or Calcein AM (623 Da) loaded into donor cells). Gap 26 (100-500 uM) is added to the culture medium prior to or during the assay. After incubation, the number of dye-coupled cells adjacent to the scratch is quantified by fluorescence microscopy. Gap 26 significantly reduces the number of dye-coupled cells, confirming inhibition of GJIC. Gap 26 also blocks ATP release via Cx43 hemichannels in ATP release assays, where cells loaded with the ATP bioluminescent reagent luciferin/luciferase show reduced luminescence upon treatment. Additionally, Gap 26 inhibits calcium waves propagating between cells via gap junctions. The TFA salt does not affect peptide activity. |
| ln Vivo |
Gap 26 TFA has been studied in vivo in various animal models to investigate the role of Cx43 gap junctions in physiology and disease. In models of spinal cord injury, intrathecal injection of Gap 26 (10-100 uM, 5-10 uL) reduces secondary injury, inflammation, and glial scar formation, and improves functional recovery. In models of cardiac ischemia/reperfusion injury, systemic administration of Gap 26 (0.5-5 mg/kg, iv) reduces infarct size and preserves cardiac function by preventing the spread of apoptotic signals through gap junctions during reperfusion. In models of epilepsy, intracerebroventricular injection of Gap 26 reduces seizure severity and propagation, suggesting that gap junction communication contributes to seizure spread. In cancer models, intratumoral injection of Gap 26 in combination with chemotherapy or immunotherapy may enhance tumor cell killing by preventing drug transfer from sensitive to resistant cells. In these models, peptide efficacy depends on the route of administration and tissue-specific Cx43 expression.
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| Enzyme Assay |
For direct binding assays of Gap 26 to Cx43, a surface plasmon resonance (SPR) or ELISA-based binding assay can be performed. A synthetic peptide corresponding to the first extracellular loop (E1) of Cx43 (residues 46-75) is immobilized on a sensor chip or 96-well plate. Varying concentrations of Gap 26 (0.1-100 uM) are flowed over the immobilized peptide. Alternatively, a pull-down assay using biotinylated Gap 26 can be used: biotin-Gap 26 (10-50 uM) is incubated with membranes from Cx43-expressing cells (e.g., cardiac myocytes, HeLa-Cx43) for 1-2 hours at 4degC. Streptavidin-agarose beads are added to capture the biotinylated peptide and any bound Cx43. Bound proteins are eluted, separated by SDS-PAGE, and immunoblotted with anti-Cx43 antibody. However, because Gap 26 is believed to block channels by interacting with the extracellular loops of intact Cx43 hemichannels rather than binding to the linear peptide in solution, traditional enzyme/receptor binding assays are less relevant. The activity of Gap 26 is assessed functionally (dye transfer, ATP release, Ca2+ wave propagation, electrical coupling) rather than by direct binding.
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| Cell Assay |
For a typical gap junction inhibition assay, cells expressing Cx43 (e.g., primary rat cardiomyocytes, HeLa cells stably transfected with Cx43, NRK-49F rat kidney epithelial cells, or U251 glioma cells) are seeded in 35 mm glass-bottom culture dishes or 24-well plates with coverslips and grown to confluence (~24-48 hours). Gap 26 TFA is reconstituted in sterile water or PBS at 5-10 mM stock and stored at -20degC. For dye transfer assays: cells are pre-incubated with Gap 26 (100-500 uM) in culture medium for 30-60 minutes at 37degC. For the scrape loading/dye transfer assay, a sterile scalpel blade or 26-gauge needle is used to create a scratch across the cell monolayer. The cells are gently washed and then incubated with the gap junction-permeable dye (e.g., Lucifer Yellow CH, 0.1-0.5 mg/mL in PBS) for 1-5 minutes at room temperature. Cells are washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, and visualized by fluorescence microscopy (excitation/emission for Lucifer Yellow: 430 nm/540 nm). The number of dye-coupled cells (fluorescent cells beyond the scratched edge) is counted in at least 5-10 microscopic fields per condition. For ATP release assay: cells are pre-incubated with Gap 26 (100-500 uM) for 30 minutes, then treated with 10 uM ionomycin or mechanical stimulation to trigger ATP release. The supernatant is collected, and ATP concentration is measured using a luciferin-luciferase bioluminescence assay (ATP Bioluminescence Assay Kit, e.g., Promega ENLITEN). Luminescence is measured in a luminometer, and ATP levels are normalized to control conditions. All treatments should be performed in triplicate wells, with at least three independent experiments.
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| Animal Protocol |
For in vivo studies, adult male Sprague-Dawley rats (250-300 g) or C57BL/6J mice (20-30 g) are used. Gap 26 TFA is reconstituted in sterile PBS or artificial CSF (for CNS studies). For intrathecal (i.t.) administration in rats: under isoflurane anesthesia, a 30-gauge needle attached to a 50-uL Hamilton syringe is inserted into the subarachnoid space between L4 and L5 vertebrae, and Gap 26 (10-100 uM in 5-10 uL volume) is injected slowly over 1 minute. For intracerebroventricular (i.c.v.) injection in mice: using a stereotaxic apparatus, a 26-gauge needle is inserted into the lateral ventricle (coordinates: -0.5 mm AP, +1.0 mm ML from bregma, -2.5 mm DV), and Gap 26 (0.5-5 nmol in 2-5 uL) is injected over 2-5 minutes. For cardiac ischemia/reperfusion studies: rats are anesthetized, and the left anterior descending coronary artery is ligated for 30-45 minutes of ischemia, followed by 2-24 hours of reperfusion. Gap 26 (0.5-5 mg/kg) is administered intravenously via the femoral vein or tail vein 5 minutes before reperfusion. At the endpoint, hearts are harvested, stained with Evans blue and TTC (triphenyltetrazolium chloride), and infarct size (as a percentage of area at risk) is measured by planimetry. For spinal cord injury studies: after laminectomy, a moderate contusion injury is induced using a weight-drop device. Immediately after injury and/or 24 hours later, Gap 26 (10-100 uM in 5 uL) is injected intrathecally. Motor function recovery is assessed using the Basso, Beattie, Bresnahan (BBB) open-field locomotor scale for 21-28 days post-injury. At sacrifice, spinal cord tissue is collected for histological analysis (lesion size, glial fibrillary acidic protein (GFAP) staining for glial scar). In all studies, control animals receive vehicle (PBS) or a scrambled Gap 26 peptide. Gap 26 is well-tolerated at the doses described, with no overt behavioral or physiological abnormalities in most studies.
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| ADME/Pharmacokinetics |
No specific pharmacokinetic data are available for Gap 26 TFA. As a 13-amino acid peptide (MW ~1.4 kDa), Gap 26 is likely to be rapidly cleared from circulation and degraded by proteases in vivo when administered systemically. Following intravenous injection, the plasma half-life of such a short peptide is expected to be on the order of a few minutes (5-15 minutes) due to rapid renal clearance (glomerular filtration) and proteolytic degradation by tissue and plasma peptidases (e.g., amino- and carboxypeptidases, endopeptidases). For CNS applications, direct intrathecal (i.t.) or intracerebroventricular (i.c.v.) injection is used to achieve local concentrations sufficient for gap junction blockade. In CSF, the peptide may have a longer half-life (30-60 minutes) due to lower protease activity compared to plasma. The TFA counterion does not affect the PK profile. Gap 26 is not intended for therapeutic use, and formal PK studies are not commonly performed.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for Gap 26 TFA. In vitro, Gap 26 (up to 500 uM) does not cause significant cytotoxicity in cultured cells (e.g., MTT or LDH release assays) over 24-48 hours of exposure. In vivo, in various animal models (spinal cord injury, cardiac ischemia, epilepsy), Gap 26 administered intrathecally, intracerebroventricularly, or intravenously at the described doses (0.5-5 mg/kg or 10-100 uM local concentration) is generally well-tolerated, with no reports of mortality, severe adverse effects, or organ toxicity. The peptide is considered to have low toxicity due to its mechanism-specific action and lack of off-target effects at concentrations used for gap junction blockade. No genotoxicity, carcinogenicity, or reproductive toxicity has been reported. The TFA salt is present in low amounts and is considered non-toxic. Gap 26 is for research use only.
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| References |
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| Additional Infomation |
Gap junctions are specialized intercellular channels that directly connect the cytoplasm of adjacent cells, allowing the passage of ions (e.g., K+, Ca2+) and small molecules (e.g., cAMP, IP3, ATP, glucose, amino acids) up to ~1 kDa, thereby enabling electrical and metabolic coupling. Connexin 43 (Cx43) is the most widely expressed and studied connexin isoform, found in many cell types. Gap 26 (sequence VCYDKSFPISHVR, residues 63-75 of Cx43 extracellular loop 1) is a synthetic peptide that selectively blocks Cx43 gap junction channels and hemichannels. It is one of a family of connexin-specific mimetic peptides, including Gap 27 (targeting Cx43 E2 loop), Gap 19 (targeting Cx43 cytoplasmic loop), and peptides specific for Cx32, Cx36, and Cx40. The TFA salt is used to improve peptide solubility and stability. Gap 26 has been widely used as a research tool to study the role of gap junction communication in cardiac electrophysiology, neurobiology, and cancer. As of 2026, no gap junction blocker has been approved for clinical use, though preclinical studies suggest potential therapeutic applications in arrhythmias, stroke, traumatic brain injury, and cancer. Gap 26 is not an approved drug; it is a research-grade biochemical reagent for laboratory use only.
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| Molecular Formula |
C72H108F3N19O21S
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| Molecular Weight |
1664.80
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| Related CAS # |
Gap 26;197250-15-0
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :~100 mg/mL (~60.07 mM)
H2O :~50 mg/mL (~30.03 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (1.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (1.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (1.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 100 mg/mL (60.07 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.6007 mL | 3.0034 mL | 6.0067 mL | |
| 5 mM | 0.1201 mL | 0.6007 mL | 1.2013 mL | |
| 10 mM | 0.0601 mL | 0.3003 mL | 0.6007 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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