| Size | Price | |
|---|---|---|
| 100mg | ||
| 250mg | ||
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Histone mark
|
|---|---|
| ln Vitro |
In this study, researchers report the evolution of an orthogonal amber suppressor pyrrolysyl-tRNA synthetase (PylRS)/tRNACUA(Pyl) pair that genetically encodes the post-translationally modified amino acid, ε-N-2-hydroxyisobutyryl-lysine (HibK), in bacteria and mammalian cells. HibK is a new type of histone mark that is widely distributed in histone proteins. The ability to site-specifically incorporate HibK into proteins provides a useful tool to probe the biological function of this newly identified post-translational modification[1].
|
| Enzyme Assay |
Determination of aaRS polyspecificity[1]
pLeiG-GFP-Asp134TAG and pBK-HibKRS-1 or pBK-HibKRS-2 plasmids were transformed into Escherichia coli DH10B. Cells were grown in GMML media, supplemented with chloramphenicol (25 μg/ml), kanamycin (25 μg/ml) and 1 mM various unnatural amino acids at 37 °C to an OD600 of 0.6, at which point IPTG was added to a final concentration of 1 mM. After 16 h expression at 30 °C, the cells were harvested by centrifugation at 4,700 ×g for 10 minutes and washed three times with PBS. Cells resuspended in PBS were transferred to a clear bottom 96 well plate and GFP-fluorescence was measured using a plate reader (485 nm excitation and 515 nm emission).[1] Expression and purification of GFP[1] pLeiG-GFP-Asp134TAG and pBK-HibKRS-1 or pBK-HibKRS-2 plasmids were transformed into Escherichia coli DH10B. Cells were grown in 2× YT media, supplemented with chloramphenicol (25 μg/ml), kanamycin (25 μg/ml) and 1 mM unnatural amino acids at 37 °C to an OD600 of 0.6, at which point IPTG was added to a final concentration of 100 μM. After 20 h expression at 30 °C, the cells were harvested by centrifugation at 4,700 ×g for 10 minutes. To purify the protein, the cell pellets were resuspended in BugBuster protein extraction reagent and lysed at 30 °C. The resulting cell lysate was clarified by centrifugation at 18,000 ×g for 30 minutes and the proteins were purified on Ni2+-NTA resin following the manufacturer’s instructions. |
| Cell Assay |
Expression and purification of Histone H2B[1]
pUltra-HibKRS-1 and pLeiG-H2B-K20HibK or pLeiG-H2B-K24HibK plasmids were transformed into Escherichia coli DH10B. Cells were grown in 2× YT media, supplemented with chloramphenicol (25 μg/ml), spectinomycin (25 μg/ml) and 1 mM unnatural amino acids at 37 °C to an OD600 of 0.6, at which point IPTG was added to a final concentration of 100 μM. After 20 h expression at 37 °C, the cells were harvested by centrifugation at 4,700 ×g for 10 minutes. To purify the protein, the cell pellets were resuspended in BugBuster protein extraction reagent and lysed at 30 °C. The resulting cell lysate was clarified by centrifugation at 18,000 ×g for 30 minutes and the proteins were purified on Ni2+-NTA resin under denaturing condition following the manufacturer’s instructions.[1] Expression and Purification of EGFP mutants in HEK293T cells[1] Adherent HEK293T cells were maintained in DMEM media supplemented with 10% heat inactivated fetal bovine serum (FBS) at 37 °C in a humidified chamber with 5% CO2. HEK293T cells were seeded in a 6-well plate at 5×105 cells/well in DMEM media, supplemented with 10% FBS and 0.5 % Antibiotic-Antimycotic. Transfection was performed with pShax-HibKCUA and pAcBac2.tR4-EGFP* using FuGENE® HD Transfection Reagent (Promega) according to manufacturer’s protocol. Cells were lysed with 250 μL CelLytic M buffer supplemented with 10 unit/mL benzonase (EMD) for 1 hour. Cell extract was clarified by centrifugation at 14,000 ×g for 3 mins and EGFP was purified by Ni2+-NTA affinity chromatography following the manufacturer’s instructions. The isolated protein was characterized by SDS-PAGE analysis followed by coomassie staining, and by ESI-MS analysis using an Agilent 1100 series LC/MS instrument. Protein concentrations were measured using Coomassie Plus (Bradford) Protein Assay kit from Pierce. |
| References |
[1]. Xiao H, et al. Genetic Incorporation of ε-N-2-Hydroxyisobutyryl-lysine into Recombinant Histones. ACS Chem Biol. 2015 Jul 17;10(7):1599-603.
|
| Additional Infomation |
N(6)-(2-hydroxyisobutanoyl)-L-lysine is an N(6)-acyl-L-lysine that has 2-hydroxyisobutanoyl as the N(6)-acyl group.
|
| Molecular Formula |
C10H20N2O4
|
|---|---|
| Molecular Weight |
232.28
|
| Exact Mass |
232.142
|
| CAS # |
1644442-22-7
|
| PubChem CID |
129031643
|
| Appearance |
Solid powder
|
| LogP |
-3
|
| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
7
|
| Heavy Atom Count |
16
|
| Complexity |
253
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
[C@@H](N)(C(O)=O)CCCCNC(=O)C(O)(C)C
|
| InChi Key |
SVPGURLIUMTWMM-ZETCQYMHSA-N
|
| InChi Code |
InChI=1S/C10H20N2O4/c1-10(2,16)9(15)12-6-4-3-5-7(11)8(13)14/h7,16H,3-6,11H2,1-2H3,(H,12,15)(H,13,14)/t7-/m0/s1
|
| Chemical Name |
(2S)-2-amino-6-[(2-hydroxy-2-methylpropanoyl)amino]hexanoic acid
|
| Synonyms |
HibK; SCHEMBL18741145; CHEBI:145391; N(6)-(2-hydroxyisobutyryl)-L-lysine; N(6)-(2-hydroxyisobutanoyl)-L-lysine;
|
| 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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| 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 | 4.3051 mL | 21.5257 mL | 43.0515 mL | |
| 5 mM | 0.8610 mL | 4.3051 mL | 8.6103 mL | |
| 10 mM | 0.4305 mL | 2.1526 mL | 4.3051 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
