Analytical grade (NH4)2CO3, CaCl2·2H2O and L -aspartic acid sodium salt monohydrate were purchased from Sigma-Aldrich and used as received. Na2SiO3 solution (1.35 g cm−3) was from Merck Chemicals, and aqueous stock solutions were prepared using Milli-Q water, 18.2 MΩcm. Stock solutions of L -α PC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, >99%, Sigma) were prepared in HPLC-grade chloroform. Glassware used to prepare solutions was soaked overnight in 10% w/v NaOH, followed by rinsing with dilute HCl and finally washing with Milli-Q water. Glass slides and crystallizing dishes were placed overnight in Piranha solution (70:30 wt% H2SO4: H2O2) and then washed copiously with Milli-Q water before drying with acetone.
L aspartic acid sodium salt monohydrate
Manufactured by Merck Group
L-aspartic acid sodium salt monohydrate is a laboratory chemical used as a component in various buffer solutions and as a precursor for the synthesis of other organic compounds. It is a white, crystalline powder that is soluble in water. This product provides the L-aspartate ion, which is a common constituent in biochemical applications.
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Lab products found in correlation
Nh4 2co3, by Merck Group (1 mentions)
Cacl2 2h2o, by Merck Group (1 mentions)
L α pc, by Merck Group (1 mentions)
Calf intestinal phosphatase, by New England Biolabs (1 mentions)
Deuterated water, by Cambridge Isotopes (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Serine, by Merck Group (1 mentions)
Phosphoenolpyruvate, by Merck Group (1 mentions)
Pspcas9 bb 2a gfp, by Addgene (1 mentions)
Galactose, by Merck Group (1 mentions)
Cell proliferation kit 2 xtt, by Merck Group (1 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
2 thenoyltrifluoroacetone, by Merck Group (1 mentions)
L glutamine solution, by Merck Group (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Glycine, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
6 protocols using l aspartic acid sodium salt monohydrate
1
Preparation of Phospholipid Monolayers
2
Purification and Characterization of Enzymes
3
CRISPR/CAS9 Silencing Assay in Cancer Cells
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143B (ATCC® CRL-8303™) and A549 (ATCC® CCL-185™) cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin at 37 °C, 5% CO2. siRNA expression vector pSilencer™ Puro Expression Vectors kit (Applied Biosystems,Life technologies), the CRISPR/CAS 9 plasmid pSpCas9(BB)-2A-GFP was from Addgene and TurboFect transfection reagent from Thermo Scientific. The kits: The quick ligation kit (NEB); RNeasy mini kit (Qiagen); High Capacity cDNA Reverse Transcription Kit (Applied Biosystems); KAPA SYBR® FAST qPCR Kit (Kapa Biosystems); Cell Proliferation Kit II (XTT) (Sigma-Aldrich); Reagents: L-glutamine solution, dialyzed FBS, glucose, galactose, glycine, serine, L-aspartic acid sodium salt monohydrate, OAA, phosphoenolpyruvate (PEP), β-nicotinamide adenine dinucleotide sodium salt and aminooxyacetate (AOA), Antimycin A and 2-thenoyltrifluoroacetone were from Sigma. The glucose and pyruvate-free and glutamine free-DMEM were from Thermo Fisher Scientific.
4
LXR Modulation and Metabolic Regulation
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ABT263, WEHI‐539, A1210477, GW3965, LXR623, ABT199, and adenosine triphosphate (ATP) were obtained from Selleckchem. Low‐density lipoprotein (LDL) from human plasma was acquired from Thermo Fisher. l ‐Aspartic acid sodium salt monohydrate was purchased from Sigma. Reagents were dissolved in dimethylsulfoxide (DMSO) (10 mM stock solution) and stored at −20°C. Final concentrations of DMSO were below 0.1% (v/v). The following antibodies were used: ABCA1 (Abcam ab18180; 1:25), Mcl‐1 (Cell Signaling Technology (CST) 5453SS; 1:500), BAK (CST 12105S; 1:500), Bcl‐2 (Abcam ab59348; 1:500), BIM (CST 2933S; 1:500), Bcl‐xL (CST 2764S; 1:500), Usp9X (CST 15751S; 1:1,000), Noxa (Millipore OP180; 1:500), β‐actin (Sigma‐Aldrich A1978, clone AC15; 1:2,000), ATF3 (CST 33593S; 1:500), ATF4 (CST 11815S; 1:500), Vinculin (Abcam ab129002; 1:200), AMPK (CST 5831S; 1:25), pAMPK (CST 2531S; 1:25), PAPRP (CST 9532S; 1:500), caspase‐9 (CST 9502S; 1:500), GRP78 (CST 3177S; 1:25), HA (CST 3724S; 1:500), LXRβ (Abcam ab56237; 1:25), SDHB (Abcam ab14714, 1:25), OXPHOS (Abcam ab110411; 1:500), and secondary goat anti‐mouse IgG‐HRP‐linked (SC2005) and secondary goat anti‐rabbit IgG‐HRP‐linked antibodies (SC2004) were purchased from Santa Cruz Biotechnology, Inc.
5
Crystallization of gpASNase(1-309)
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A variety of crystallization screens
(Qiagen) were applied to purified His6-TEV-gpASNase(1–309).
Several hits were obtained that contained buffers ranging from pH
5.0–6.5, 0.1–0.2 M salt (ammonium acetate, ammonium
sulfate, lithium sulfate), and 17–25% PEG 3350–10000.
Condition 22 from The Protein Complex Suite (0.15 M ammonium sulfate,
0.1 M MES at pH 6.0, and 15% PEG 4000) was optimized. For setups,
1 μL of protein (35–45 mg/mL in 20 mM CAPS at pH 10.5,
100 mM KCl, and 2 mM DTT) was mixed with 1 μL of reservoir solution
(0.2 M ammonium sulfate, 0.1 M MES at pH 6.0, and 13–15% PEG
3350) on a glass coverslip and left to undergo vapor diffusion using
the hanging drop method at 20 °C. Large rod-like crystals grew
within 1 day. To obtain the Asp-bound gpASNase3 complex, crystals
were first soaked in a solution (0.1 M MES at pH 6.0 and 15% PEG 3350)
containing 2 M glycine to cleave the enzyme followed by a soak in
cryoprotectant solution (0.1 M MES at pH 6.0, 15% PEG 3350, and 30%
ethylene glycol) containing saturatedl -aspartic acid sodium
salt monohydrate (Sigma A6683).
(Qiagen) were applied to purified His6-TEV-gpASNase(1–309).
Several hits were obtained that contained buffers ranging from pH
5.0–6.5, 0.1–0.2 M salt (ammonium acetate, ammonium
sulfate, lithium sulfate), and 17–25% PEG 3350–10000.
Condition 22 from The Protein Complex Suite (0.15 M ammonium sulfate,
0.1 M MES at pH 6.0, and 15% PEG 4000) was optimized. For setups,
1 μL of protein (35–45 mg/mL in 20 mM CAPS at pH 10.5,
100 mM KCl, and 2 mM DTT) was mixed with 1 μL of reservoir solution
(0.2 M ammonium sulfate, 0.1 M MES at pH 6.0, and 13–15% PEG
3350) on a glass coverslip and left to undergo vapor diffusion using
the hanging drop method at 20 °C. Large rod-like crystals grew
within 1 day. To obtain the Asp-bound gpASNase3 complex, crystals
were first soaked in a solution (0.1 M MES at pH 6.0 and 15% PEG 3350)
containing 2 M glycine to cleave the enzyme followed by a soak in
cryoprotectant solution (0.1 M MES at pH 6.0, 15% PEG 3350, and 30%
ethylene glycol) containing saturated
salt monohydrate (Sigma A6683).
6
Purification and Characterization of LiAS-A
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L-asparagine, L-aspartic acid sodium salt monohydrate, L-glutamine, L-glutamatic acid salt hydrate, ATP disodium salt hydrate, AMP disodium salt, sodium pyrophosphate decahydrate, ninhydrin, dNTPs, ammonium chloride, magnesium chloride, tween-20, tris-base, urea, thiourea, DTT, triton X-100 and IPTG (isopropyl-β-D- thiogalactopyranoside) were purchased from Sigma. Oligonucleotide primers were obtained from STAB VIDA. Restriction endonucleases were from New England Biolabs. Polyclonal antibodies against LiAS-A were obtained in rabbits inoculated with purified recombinant His-tagged LiAS-A. E. coli L-asparaginase was purchased from Prospec.
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