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| Targets |
In plants, Flagelin 22 targets the FLS2 receptor (flagellin-sensitive 2), a leucine-rich repeat receptor-like kinase (LRR-RLK) that recognizes a conserved 22-amino acid epitope (flg22) of bacterial flagellin. FLS2 activation initiates a signaling cascade involving BAK1 (BRI1-associated receptor kinase 1), MAPK cascades, calcium influx, and transcriptional reprogramming leading to plant innate immunity. In algae, Flagelin 22 may activate similar defense mechanisms.
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| ln Vitro |
Flagelin 22, or FLG22, is a 22-amino-acid peptide that can trigger an immune response in a variety of plants, including tobacco (Nicotiana tabacum), potato (Solanum tuberosum), tomato (Solanum lycopersicum), and Arabidopsis thaliana. It is a 22-amino-acid peptide that matches to the highly conserved N-terminal portion of flagellin. Saccharina japonica female gametophytes and sporophytes are both capable of inducing oxidative bursts and hypersensitive reactions (HR) in response to flagelin 22, suggesting that plants and algae may have comparable pathogen recognition systems.Following a 40-day incubation period in the presence of Flagelin 22, flg15, flg14, and flg22D43A, the female gametophytes of S. japonica showed a significant growth inhibition response to both Flagelin 22 and flg15 at a concentration of 1 μM. Female gametophytes challenged with flagelin 22 and flagelin 15 had fresh weights that are less than half of the control[1].
In vitro, Flagelin 22 (flg22) at concentrations of 1-100 uM triggers defense responses in plant cell cultures. In Nicotiana tabacum, Solanum tuberosum, Solanum lycopersicum, and Arabidopsis thaliana, flg22 induces oxidative burst (reactive oxygen species production), callose deposition, and expression of defense-related genes. It also inhibits plant growth at higher concentrations. In Saccharina japonica (brown alga), flg22 at 1 uM induces significant growth inhibition, oxidative burst, and hypersensitive response (HR)-like reactions in both female gametophytes and sporophytes after a 40-day latent period. Flg15, flg14, and flg22D43A show weaker or no activity, confirming the specificity of the flg22 epitope. |
| ln Vivo |
In vivo, Flagelin 22 is used as an immune elicitor in plant research. Application of flg22 to Arabidopsis leaves triggers systemic acquired resistance (SAR) and protects against subsequent infection by pathogenic bacteria such as Pseudomonas syringae pv. tomato DC3000. It is typically applied by infiltration (1-10 uM) or spray onto leaves. In algae such as Saccharina japonica, flg22 (1 uM) applied to cultures induces defense responses and growth inhibition over extended periods (up to 40 days). Flagelin 22 is also used in plant cell suspension cultures to study early signaling events (e.g., calcium influx, MAPK activation). For in planta assays, flg22 is injected into the apoplast of leaves using a needleless syringe. Treated leaves are harvested at time points ranging from minutes (oxidative burst, MAPK phosphorylation) to hours (gene expression) to days (callose deposition, growth inhibition).
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| Enzyme Assay |
Cell-free assays are not applicable as flg22 acts through a plant receptor in living cells. For receptor binding studies using plant membrane fractions: Isolate microsomal membrane fractions from Arabidopsis leaves expressing FLS2. Incubate membranes (50 ug) with biotinylated flg22 (10-100 nM) in binding buffer (25 mM MES-KOH, pH 5.7, 5 mM KCl, 1 mM CaCl2, 10 mM MgCl2, 0.1% BSA) for 30-60 minutes at 4degC. Add streptavidin-agarose beads, incubate, wash, and elute bound proteins. Separate by SDS-PAGE and detect FLS2 by immunoblotting. For competition assays, add unlabeled flg22 (1 nM-100 uM) to determine binding affinity. For MAPK phosphorylation assays (cell-free leaf extracts), treat Arabidopsis leaf disks with flg22 (1 uM) for 0-30 minutes, flash-freeze, grind in liquid nitrogen, extract protein, and perform Western blot with anti-phospho-p44/42 MAPK antibody (which recognizes plant MAPKs like MPK3 and MPK6).
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| Cell Assay |
For plant cell culture assays, grow Arabidopsis thaliana cell suspension cultures (or leaf protoplasts) in Murashige and Skoog (MS) medium. Treat cells with Flagelin 22 TFA at concentrations ranging from 10 nM to 10 uM for 5-30 minutes (for oxidative burst) to 24 hours (for gene expression). Measure oxidative burst (reactive oxygen species) by adding luminol (100 uM) and peroxidase (20 ug/mL) to the culture medium and measuring chemiluminescence immediately after flg22 addition. For callose deposition assays, infiltrate Arabidopsis leaves with flg22 (1 uM) using a needleless syringe. After 12-24 hours, clear leaves with 95% ethanol, stain with aniline blue (0.01% in 150 mM K2HPO4, pH 9.5), and visualize callose deposits as yellow-green fluorescent spots under UV light (excitation 365 nm, emission 420 nm). Quantify callose deposits per mm2 leaf area using image analysis software. For gene expression analysis, treat cells or leaf disks with flg22 (1 uM) for 0-24 hours, extract RNA, reverse transcribe, and perform qPCR for defense marker genes such as FRK1 (Flg22-induced receptor-like kinase 1), WRKY transcription factors, and PR1 (pathogenesis-related protein 1). Normalize to housekeeping genes (e.g., ACTIN2 or UBQ10). For MAPK activation assays, treat leaf disks with flg22 (100 nM-1 uM) for 0-30 minutes, extract protein, and perform Western blot using anti-phospho-p44/42 MAPK antibody. For growth inhibition assays, grow Arabidopsis seedlings in liquid MS medium with 0.5% sucrose in 24-well plates, add flg22 (1 uM) or control, measure fresh weight after 7-14 days. For studies in algae, culture Saccharina japonica female gametophytes in Provasoli-enriched seawater. Add flg22 (1 uM) or control peptides (flg15, flg14, flg22D43A). After 40 days of latent period, measure growth by fresh weight and assess oxidative burst using nitroblue tetrazolium (NBT) staining for superoxide and trypan blue staining for cell death (hypersensitive response).
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| Animal Protocol |
For in planta assays, use Arabidopsis thaliana (ecotype Col-0) or other plant species (tobacco, tomato, potato). Grow plants under short-day (8-10 h light) or long-day (16 h light) conditions at 22degC for 4-6 weeks. Prepare Flagelin 22 TFA solution in water (or 0.05% Silwet L-77 as surfactant) at 1-10 uM for infiltration. For immune activation, infiltrate leaves with flg22 using a needleless syringe (for Arabidopsis, inject into the abaxial side of fully expanded leaves). For systemic immunity studies, treat only the lower leaves (3-4 leaves per plant). At various time points after treatment (0.5, 1, 2, 4, 8, 12, 24, 48 h), harvest treated or distant (untreated) leaves. For oxidative burst assays, take leaf disks (6 mm diameter) immediately after infiltration and measure ROS as described for cell cultures. For callose deposition, stain and quantify after 12-24 h. For bacterial infection assays, challenge plants with Pseudomonas syringae pv. tomato DC3000 (OD600=0.0002-0.002) by infiltration 24 hours after flg22 treatment. After 3 days, harvest leaf disks, homogenize, plate serial dilutions on selective media, and count colony-forming units (CFU) to assess bacterial growth. For gene expression analysis, freeze leaf samples in liquid nitrogen, grind, extract RNA, and perform qPCR. For growth inhibition, spray seedlings with flg22 (1 uM) daily for 7-10 days and measure rosette diameter and fresh weight. For MAPK activation in vivo, treat leaves through petioles with flg22 solution and harvest tissue at 0, 5, 15, 30, 60 minutes. For algae experiments, culture Saccharina japonica female gametophytes in flasks with Provasoli-enriched seawater. Add flg22 at 1 uM and control peptides (flg15, flg14, flg22D43A) for up to 40 days. Change medium weekly. Measure growth (fresh weight), oxidative burst (NBT staining), and cell death (trypan blue staining).
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| ADME/Pharmacokinetics |
Flagelin 22 TFA has a molecular weight of 2386.50 and formula C95H163F3N32O36. The TFA salt enhances water solubility. In plants, flg22 is applied at 1-10 uM for most assays. The peptide is stable in aqueous solution for short-term use at 4degC and should be stored as powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month). Protect from light and moisture. For in vitro use, dissolve in water or 10 mM MES-KOH buffer (pH 5.7) to prepare stock solutions (1-10 mM). For in planta infiltration, dilute in water to working concentration (1-10 uM). The TFA counterion may affect plant growth at very high concentrations; control samples should include equivalent TFA concentrations. The peptide corresponds to the N-terminal region of flagellin (first 22 amino acids: QRLSTGSRINSAKDDAAGLQIA). The sequence is highly conserved among many bacterial species. For research use only; not for human or veterinary applications.
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| Toxicity/Toxicokinetics |
Flagelin 22 TFA is generally considered safe for laboratory use under standard chemical safety guidelines. The compound is a peptide and is not toxic to mammals at the concentrations used in plant research (1-10 uM). It is not intended for human consumption. The TFA salt may cause mild irritation to eyes, skin, and respiratory tract. Standard laboratory precautions (gloves, lab coat, safety glasses) should be used. Avoid inhalation of fine powder. Dispose of waste according to institutional guidelines. In case of accidental exposure, wash affected area with soap and water. For eye exposure, rinse with water for 15 minutes. The compound is not classified as hazardous for transport under UN regulations. No genotoxicity or carcinogenicity data are available. For large-scale use, consult safety data sheet (SDS).
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| References |
[1]. Bojun Lu, et al. Defense responses in female gametophytes of Saccharina japonica (Phaeophyta) induced by flg22-derived peptides. Journal of Applied Phycology (2016), 28(3), 1793-1801.
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| Additional Infomation |
Flagelin 22 TFA (flg22) is a 22-amino acid peptide derived from the conserved N-terminus of bacterial flagellin. It is a well-characterized microbe-associated molecular pattern (MAMP) that triggers pattern-triggered immunity (PTI) in plants and algae. The peptide is recognized by the FLS2 receptor kinase, leading to a cascade of defense responses including MAPK activation, oxidative burst, callose deposition, and induction of defense gene expression. Flg22 is widely used as a positive control in plant immunity research, for screening for PTI-deficient mutants, and for studying plant-pathogen interactions. The TFA salt form enhances solubility. The peptide is for research use only and not for diagnostic or therapeutic applications.
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| Molecular Formula |
C95H163F3N32O36
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| Molecular Weight |
2386.50
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| Related CAS # |
Flagelin 22;304642-91-9
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| Appearance |
Typically exists as solid at room temperature
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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 |
| 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 (~41.90 mM)
H2O :~33.33 mg/mL (~13.97 mM) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.4190 mL | 2.0951 mL | 4.1902 mL | |
| 5 mM | 0.0838 mL | 0.4190 mL | 0.8380 mL | |
| 10 mM | 0.0419 mL | 0.2095 mL | 0.4190 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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