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| ln Vitro |
Advice (This is our suggested protocol, which should be adjusted to suit your particular circumstances as it simply offers guidance). CypK assay (antibody expression): 1. In a 250 mL flask with 50 mL of expression medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B, thaw a vial of HEK suspension cells. The cells should be kept in a humidified incubator with a 125 rpm shaker, 37 °C, and 8% CO2. Before transfection, divide cells at least twice to 0.3–0.5 x 106 cells/mL (every 2-3 days). 2. After two to three days of splitting, when the density of 2.5 x 106 cells/mL is attained, make a new solution of 100 mM CypK. Weigh 64 mg of CypK, add 2.5 mL of 0.1 NaOH, vortex, spin to collect any remaining particles, and then sonicate to accomplish this. 3. Pour 2.5 mL of CypK (100 mM in 0.1 M NaOH) into 42.5 mL of antibiotic-supplemented expression medium. Mix well, add 250 µL of 0.1 M HCl, and sterilize using a 0.22 µm filter. 4. Dilute 50 µg of HC and LC pKym1 plasmid to 2.5 mL in reduced serum medium. In a separate tube, dilute 135 µL of transfection reagent to 2.5 mL with reduced serum medium. 5. To enable the formation of a complex between the DNA and transfection reagent, mix the plasmid and transfection reagent solutions and incubate for 20 minutes after preparing the solution for 5 minutes. 6. In the meantime, resuspend 125 million cells in expression medium containing CypK, centrifuge for 5 minutes at 500 xg, and add the DNA transfection reagent mix. 7. After incubating the cells for 20 hours, add 250 µL of the transfection reagent enhancer included in the kit. 8. Harvest antibodies from the supernatant 6-7 days after adding CypK (no need to change medium during expression).
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| References |
[1]. Oller-Salvia B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction. J Vis Exp. 2018 Sep 14;(139):58066.
[2]. Oller-Salvia B, et, al. Rapid and Efficient Generation of Stable Antibody-Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels-Alder Reaction. Angew Chem Int Ed Engl. 2018 Mar 5;57(11):2831-2834. |
| Molecular Formula |
C12H20N2O4
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|---|---|
| Molecular Weight |
256.298203468323
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| Exact Mass |
256.142
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| CAS # |
1610703-09-7
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| Appearance |
Solid powder
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| LogP |
-2
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| SMILES |
O(C(NCCCCC(C(=O)O)N)=O)CC1C=C1C
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| InChi Key |
YMBQDEOCKOLCJG-AXDSSHIGSA-N
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| InChi Code |
InChI=1S/C12H20N2O4/c1-8-6-9(8)7-18-12(17)14-5-3-2-4-10(13)11(15)16/h6,9-10H,2-5,7,13H2,1H3,(H,14,17)(H,15,16)/t9?,10-/m0/s1
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| Chemical Name |
(2S)-2-amino-6-[(2-methylcycloprop-2-en-1-yl)methoxycarbonylamino]hexanoic acid
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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 : ~30 mg/mL (~117.05 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (11.71 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 30.0 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: ≥ 3 mg/mL (11.71 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 30.0 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: ≥ 3 mg/mL (11.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.9017 mL | 19.5084 mL | 39.0168 mL | |
| 5 mM | 0.7803 mL | 3.9017 mL | 7.8034 mL | |
| 10 mM | 0.3902 mL | 1.9508 mL | 3.9017 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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