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| Other Sizes |
Purity: ≥98%
| ln Vitro |
A21960 inhibited resolvase-mediated site-specific recombination in vitro with a 50% inhibition point at approximately 1 mg/ml and complete inhibition at 5 mg/ml, as assessed using the model cointegrate plasmid πres^2 and purified resolvase protein. [1]
A21960 did not inhibit EcoRI cleavage, indicating specificity for resolvase. [1] In nitrocellulose filter binding assays and DNase I footprinting assays, A21960 did not block resolvase binding to the res site even at concentrations that completely inhibited recombination. [1] A21960 inhibited the formation of resolvase-res synaptic complexes (α-type structures) as detected by glutaraldehyde crosslinking and BamHI cleavage. [1] In topoisomerase I relaxation experiments, A21960 caused a decrease in the linking number difference (ΔLk) protected by resolvase: at 1 mg/ml and 2 mg/ml, ΔLk was -2 (compared to -3 in the absence of inhibitor), and at 3 mg/ml, ΔLk was -1, indicating that synapsis was blocked but resolvase binding and DNA wrapping at unsynapsed res sites still occurred, protecting one negative supercoil. [1] |
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| ln Vivo |
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| Enzyme Assay |
The standard resolvase recombination reaction was performed at 37°C for 15 min in a buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 1 mM dithiothreitol, 1% DMSO, 0.4 μg of πres^2 plasmid DNA, and 2 μg of resolvase. Inhibitor A21960 was preincubated with resolvase at 4°C for 1 min before adding DNA. Reactions were stopped and analyzed by agarose gel electrophoresis. [1]
Glutaraldehyde crosslinking assays to detect synaptic complexes: Resolvase was allowed to bind to res^2 DNA for 5 min at 15°C in buffer containing 150 mM NaCl, 20 mM triethanolamine-HCl (pH 7.5), and 5 mM EDTA. Freshly diluted 8% glutaraldehyde was added to a final concentration of 0.2% and incubated for 5 min at 15°C. Complexes were purified by filtration through Sepharose CL-4B minicolumns and then cleaved with BamHI for 10 min at 37°C. Products were electrophoresed through 0.8% agarose gels. [1] Topoisomerase I relaxation experiments: Glutaraldehyde-fixed synaptic complexes were treated with wheat germ topoisomerase I for 15 min at 15°C. Reactions were stopped by adding SDS to 0.2% final volume. Samples were electrophoresed through 1.2% agarose gels in TBE buffer containing 1.2 μg chloroquine/ml. [1] |
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| Animal Protocol |
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| References |
J Mol Biol.1989 Mar 20;206(2):295-304.
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| Additional Infomation |
A21960 was a generous gift from Abbott Laboratories. [1] It acts by blocking synapse formation between res sites without affecting resolvase binding or DNA bending. [1]
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| Molecular Formula |
C6H9CLO3
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|---|---|---|
| Molecular Weight |
164.5869
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| Exact Mass |
164.024
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| CAS # |
609-15-4
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| Related CAS # |
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| PubChem CID |
11858
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| Appearance |
Colorless to light yellow liquid（Density：1.172 g/cm³）
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
225.5±0.0 °C at 760 mmHg
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| Melting Point |
-80 °C
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| Flash Point |
50.0±0.0 °C
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| Vapour Pressure |
0.1±0.4 mmHg at 25°C
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| Index of Refraction |
1.434
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| LogP |
1.66
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
10
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| Complexity |
144
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC([H])(C(C([H])([H])[H])=O)C(=O)OC([H])([H])C([H])([H])[H]
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| InChi Key |
RDULEYWUGKOCMR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C6H9ClO3/c1-3-10-6(9)5(7)4(2)8/h5H,3H2,1-2H3
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| Chemical Name |
ethyl 2-chloro-3-oxobutanoate
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 6.0757 mL | 30.3785 mL | 60.7570 mL | |
| 5 mM | 1.2151 mL | 6.0757 mL | 12.1514 mL | |
| 10 mM | 0.6076 mL | 3.0379 mL | 6.0757 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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