| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| 100mg | |||
| 250mg | |||
| Other Sizes |
| Targets |
Marker dye
Hoechst 33258 analog 6 targets DNA, specifically binding to the minor groove of double-stranded DNA. The compound exhibits a preference for A/T-rich regions of DNA. As a member of the Hoechst family of bis-benzimides, the analog binds to nucleic acids through non-covalent interactions in the minor groove. This binding is characterized by high affinity and selectivity for DNA over RNA. The compound's fluorescence properties are enhanced upon binding to DNA, making it useful for visualizing nuclear DNA in live or fixed cells. The analog is designed to have enhanced DNA-binding affinity and selectivity compared to the parent compound. Its primary application is as a fluorescent stain for DNA in various research applications. |
|---|---|
| ln Vitro |
Preparation of Hoechst working slution:
1.1 Preparation of stock solution Prepare 1 mg/mL Hoechst stock solution with DMSO. Note: Hoechst stock solution is recommended to be aliquoted and stored in the dark at -4 ℃ or -20 ℃. 1.2 Preparation of working slution Dilute the stock solution with preheated serum-free cell culture medium or PBS to a final concentration of 10 μ g/mL Hoechst working solution. Note: Please adjust the concentration of Hoechst working solution according to your specific needs, and use freshly prepared solutions. 2. Cell staining (suspended cells) 2.1 Centrifuge and collect cells, wash twice with PBS for 5 minutes each time. Cell density is 1 × 10~6/mL 2.2 Add 1 mL of Hoechst working solution and incubate at room temperature for 3-10 minutes. 2.3 400 g, centrifuge for 3-4 minutes, discard the supernatant. 2.4 Wash the cells twice with PBS, each time for 5 minutes. After resuspending cells in 1 mL serum-free medium or PBS, observe them using a fluorescence microscope or flow cytometer. 3. Cell staining (adherent cells) 3.1 Cultivate adherent cells on sterile coverslips. 3.2 Remove the cover glass from the culture medium and aspirate excess culture medium. 3.3 Add 100 μ L of dye working solution, gently shake to completely cover the cells, and incubate for 3-10 minutes. 3.4 Remove the dye working solution, wash 2-3 times with culture medium for 5 minutes each time, and observe using a fluorescence microscope or flow cytometer. Hoechst 33258 analog 6 demonstrates potent in vitro DNA-binding activity. The compound binds to DNA in the minor groove with high affinity, exhibiting a preference for A/T-rich sequences. Upon binding to DNA, the compound's fluorescence is significantly enhanced, allowing for sensitive detection of nuclear DNA. The analog is designed to have enhanced DNA-binding affinity, selectivity, or fluorescence properties compared to the parent Hoechst 33258. In cell-based assays, the compound is cell-permeable and can stain DNA in live or fixed cells. Its enhanced signal and lower toxicity make it suitable for long-term live-cell nuclear imaging. The compound's activity is concentration-dependent, with optimal staining observed at appropriate concentrations. |
| ln Vivo |
In vivo activity of Hoechst 33258 analog 6 is not applicable, as the compound is a fluorescent DNA stain used primarily for in vitro and ex vivo applications. The compound is not a therapeutic agent and is not intended for in vivo administration. However, the dye can be used in ex vivo applications such as staining tissues or cells isolated from animals. The compound's cell-permeability allows it to stain DNA in live cells, making it useful for various biological studies. Its enhanced signal and lower toxicity make it suitable for long-term live-cell imaging applications. The compound is used in fluorescence microscopy, flow cytometry, and nucleic acid staining assays.
|
| Enzyme Assay |
DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions[1].
In vitro DNA binding assays for Hoechst 33258 analog 6 involve measuring the compound's binding affinity to DNA using fluorescence spectroscopy. The compound's fluorescence is significantly enhanced upon binding to DNA, allowing for sensitive detection. Binding affinity can be determined by titrating the compound with increasing concentrations of DNA and measuring the change in fluorescence. The preference for A/T-rich sequences can be assessed using different DNA sequences. Competitive binding assays can be performed using known DNA-binding compounds. The compound's purity (≥98%) is confirmed by HPLC and NMR analysis. The molecular weight of 536.71 g/mol and formula C33H40N6O have been characterized. |
| Cell Assay |
In vitro cellular assays for Hoechst 33258 analog 6 are performed using various cell lines to assess DNA staining properties. Cells are cultured in appropriate medium and incubated with varying concentrations of the compound for defined time periods. Nuclear staining is visualized using fluorescence microscopy, and fluorescence intensity is measured using flow cytometry or a fluorescence plate reader. The compound's cell-permeability allows for staining of live cells. Staining can be performed on live or fixed cells. The compound's enhanced signal and lower toxicity make it suitable for long-term live-cell imaging. Cytotoxicity is assessed in parallel using standard viability assays to ensure that staining does not affect cell viability. Staining specificity is confirmed using DNase treatment or competition with unlabeled DNA-binding compounds.
|
| Animal Protocol |
In vivo animal studies are not applicable for Hoechst 33258 analog 6, as the compound is a fluorescent DNA stain used for in vitro and ex vivo applications rather than a therapeutic agent. The compound is not intended for in vivo administration. However, the dye can be used in ex vivo applications such as staining tissues or cells isolated from animals for histological analysis. If used in vivo, standard procedures for fluorescent dyes would include monitoring for signs of toxicity. The compound's suitability for long-term live-cell imaging suggests low toxicity. Comprehensive toxicological characterization has not been reported, as the compound is intended for research use only.
|
| ADME/Pharmacokinetics |
Pharmacokinetic properties of Hoechst 33258 analog 6 are not relevant, as the compound is a fluorescent DNA stain used for in vitro and ex vivo applications rather than a therapeutic agent. The compound has a molecular formula of C33H40N6O and a molecular weight of 536.71 g/mol. It is cell-permeable, allowing it to enter live cells and stain nuclear DNA. The compound is soluble in DMSO and other organic solvents. Comprehensive pharmacokinetic parameters have not been characterized, as the compound is not intended for in vivo administration. Its stability in solution and under various storage conditions has been characterized. The compound's properties support its use in fluorescence microscopy and flow cytometry applications.
|
| Toxicity/Toxicokinetics |
Hoechst 33258 analog 6 is not a therapeutic agent and has not undergone toxicology testing for therapeutic applications. As a fluorescent DNA stain, the compound is intended for in vitro and ex vivo research use only. The compound is designed to have lower toxicity compared to the parent Hoechst 33258, making it suitable for long-term live-cell nuclear imaging. Standard in vitro cytotoxicity assays in cell lines are typically performed to ensure that staining does not affect cell viability. The compound's cell-permeability and DNA-binding properties are the primary considerations for its use. Comprehensive toxicological characterization including genotoxicity has not been reported, as the compound is not intended for human use. Hoechst 33258 analog 6 is strictly intended for research purposes.
|
| References | |
| Additional Infomation |
This article describes a variety of applications of fluorescence microscopy for detecting deoxyribonucleic acid synthesis. These applications include: (a) analyzing the exchange and separation of sister chromatids during mitosis; (b) locating regions on chromosomes containing deoxyribonucleic acid and with asymmetrical distribution of thymine residues between polynucleotide chains; and (c) detecting regions in metaphase chromosomes during late replication. In both fixed cytological specimens and unfixed cultured cells, it was confirmed that the biosynthesized 5-bromodeoxyuridine incorporated into the interphase nucleus could inhibit Hoechst 33258 fluorescence. Many of the cytological observations described in this article may lay the foundation for future biochemical research. [3]
Hoechst 33258 analog 6 is a refined variant of the Hoechst family of blue fluorescent DNA dyes. It is a cell-permeable, live-cell nuclear labeling dye that binds to the minor groove of DNA with a preference for A/T-rich sequences. The analog is designed to enhance DNA-binding affinity, selectivity, or fluorescence properties. It is suitable for long-term live-cell nuclear imaging with enhanced signal and lower toxicity. The compound has a molecular formula of C33H40N6O and a molecular weight of 536.71 g/mol. Hoechst 33258 analog 6 is used in fluorescence microscopy, flow cytometry, and nucleic acid staining assays. It has not entered clinical trials and is available for research purposes only. |
| Molecular Formula |
C33H40N6O
|
|---|---|
| Molecular Weight |
536.7103
|
| Exact Mass |
536.326
|
| CAS # |
129244-66-2
|
| Related CAS # |
Hoechst 33258;23491-44-3;Hoechst 33258 analog;258843-62-8
|
| PubChem CID |
136218783
|
| Appearance |
Off-white to pink solid powder
|
| LogP |
5.753
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
40
|
| Complexity |
838
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
LXRWWFIBSFWADZ-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C33H40N6O/c1-32(2,3)23-16-21(17-24(29(23)40)33(4,5)6)31-35-25-10-8-20(18-27(25)36-31)30-34-26-11-9-22(19-28(26)37-30)39-14-12-38(7)13-15-39/h8-11,16-19,40H,12-15H2,1-7H3,(H,34,37)(H,35,36)
|
| Chemical Name |
2,6-ditert-butyl-4-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]phenol
|
| Synonyms |
2,6-ditert-butyl-4-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]phenol
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~46.58 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.66 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 (4.66 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8632 mL | 9.3160 mL | 18.6320 mL | |
| 5 mM | 0.3726 mL | 1.8632 mL | 3.7264 mL | |
| 10 mM | 0.1863 mL | 0.9316 mL | 1.8632 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
