To obtain scFv surface expression on yeast cells, 1116NS19.9-scFv-pETCON2 or 5b1-scFv-pETCON2 transfected yeast cells were cultured in SD-Trp a synthetic defined media (SD) lacking Tryptophan (Trp) [2% glucose (Sigma), 0.67% yeast nitrogen base w/o amino acids (BD), 0.54% Na2HPO4 (Sigma), 0.86% NaH2PO4 (Sigma) and 0.192% yeast synthetic drop-out medium supplements without Trp (Sigma)] at 30 °C, passaged 1:10 each day for three days, then scFv was expressed by changing the media to SG-Trp a synthetic galactose (SG) based media [2% galactose (Sigma), 0.2% glucose, 0.67% yeast nitrogen base w/o amino acids, 0.54% Na2HPO4, 0.86% NaH2PO4, and 0.192% yeast synthetic drop-out medium supplements without Trp] and the temperature to 20 °C, and cells were grown overnight to obtain scFv-YSD cells.
Na2hpo4
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, India, France, Switzerland, China, Macao, Australia, Poland, Sao Tome and Principe, Czechia, Brazil, Canada, Singapore
Na2HPO4 is a chemical compound commonly used as a laboratory reagent. It is a white, crystalline solid with the chemical formula Na2HPO4. Na2HPO4 is a salt of sodium and phosphoric acid, and it serves as a buffer solution, maintaining a specific pH level in aqueous environments.
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529 protocols using na2hpo4
1
Yeast Surface Display of scFv
2
Fluorescent Thiol Sensing Assay
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SSP4 (3',6'-Di(O-thiosalicyl)fluorescein), Na2S2, Na2S3 and Na2S4 were purchased from Dojindo molecular Technologies Inc. (Rockville, MD). Thioredoxin was purchased from ThermoFisher Scientific (Grand Island, NY). Carbon monoxide (CO, 1 mM), carbonyl sulfide (COS, 20 mM) and sulfur dioxide (SO2, 1.4 M) solutions were prepared by bubbling pure gas through a sintered glass aerator into buffer for 20–30 min. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Phosphate buffer (in mM): 137 NaCl, 2.7, KCl, 8 Na2HPO4, 2 NaH2PO4, pH 7.4.
Sorensen's buffer in (mM): 200 Na2HPO4, 200 NaH2PO4, ratio sdjusted to pH 6, 7 or 8.
Phosphate buffer (in mM): 137 NaCl, 2.7, KCl, 8 Na2HPO4, 2 NaH2PO4, pH 7.4.
Sorensen's buffer in (mM): 200 Na2HPO4, 200 NaH2PO4, ratio sdjusted to pH 6, 7 or 8.
3
Plasma Total Antioxidant Capacity
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The total antioxidant status (TAOS) in plasma was measured by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay, which was based on the capacity of plasma to scavenge the ABTS+ radical [40 (link)]. Briefly, the relative inhibition of ABTS+ formation, after the plasma addition, is proportional to the antioxidant capacity of the sample. For the measurement, plasma was diluted with 180 µL phosphate buffer (0.076 M NaH2PO4 (POCH, Gliwice, Poland)+ 0.23 M Na2HPO4 (Sigma-Aldrich, Burlington, MA, USA) in pure water), and then it was incubated for 10 min at room temperature in a 96-well plate with a 5 µL reaction mixture containing 7 mM ABTS (Sigma-Aldrich, Burlington, MA, USA) and 2.45 mM potassium persulfate (Sigma-Aldrich, Burlington, MA, USA) (in phosphate buffer: 0.22 M NaH2PO4 (POCH, Gliwice, Poland) + 0.37 M Na2HPO4 (Sigma-Aldrich, Burlington, MA, USA)) solved in pure water. The absorbance in the test and control samples (saline instead of plasma) was read at 630 nm. Results expressed as percentage inhibition of the reaction were calculated as follows: TAOS [%] = 100 × (Ac − At)/Ac, where Ac is the absorbance of the control sample absorbance, and At is the test sample absorbance.
4
Yeast Surface Display of scFv
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Cloning of antibodies into yeast surface display (YSD) system and functional assay. Sequences of the variable domains (VH and VL) of the anti-SLe a mouse antibody 1116NS19.9 [20] (link) and the human antibody 5b1 [22] (link) were used to design scFv of (N'- Induction of scFv expression on YSD system. To obtain scFv surface expression on yeast cells, 1116NS19.9-scFv-pETCON2 or 5b1-scFv-pETCON2 transfected yeast cells were cultured in SD-Trp a synthetic defined media (SD) lacking Tryptophan (Trp)
[2% glucose (Sigma), 0.67% yeast nitrogen base w/o amino acids (BD), 0.54% Na2HPO4 (Sigma), 0.86% NaH2PO4 (Sigma) and 0.192% yeast synthetic drop-out preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
medium supplements without Trp (Sigma)] at °C, passaged 1:10 each day for three days, then scFv was expressed by changing the media to SG-Trp a synthetic galactose (SG) based media [2% galactose (Sigma), 0.2% glucose, 0.67% yeast nitrogen base w/o amino acids, 0.54% Na2HPO4, 0.86% NaH2PO4, and 0.192% yeast synthetic drop-out medium supplements without Trp] and the temperature to 20 °C, and cells were grown overnight to obtain scFv-YSD cells.
[2% glucose (Sigma), 0.67% yeast nitrogen base w/o amino acids (BD), 0.54% Na2HPO4 (Sigma), 0.86% NaH2PO4 (Sigma) and 0.192% yeast synthetic drop-out preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
medium supplements without Trp (Sigma)] at °C, passaged 1:10 each day for three days, then scFv was expressed by changing the media to SG-Trp a synthetic galactose (SG) based media [2% galactose (Sigma), 0.2% glucose, 0.67% yeast nitrogen base w/o amino acids, 0.54% Na2HPO4, 0.86% NaH2PO4, and 0.192% yeast synthetic drop-out medium supplements without Trp] and the temperature to 20 °C, and cells were grown overnight to obtain scFv-YSD cells.
5
Insulin-Protamine Binding Characterization
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Insulin human recombinant, expressed
in yeast (≥98% purity,
MW 5.8 kDa), protamine sulfate salt from salmon (MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR)
(MW 5.1 kDa), 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS),
and zinc sulfate monohydrate (ZnSO4) were purchased from
Sigma-Aldrich. Na2HPO4, NaH2PO4, NaCl, and phenol were purchased from Merck Chemicals Ltd.
All experiments were performed in phosphate buffer at pH 8.0 containing
10 mM Na2HPO4, 10 mM NaH2PO4, and NaCl. The concentration of insulin was measured using an absorbance
of 276 nm on the Cary 100 UV–vis spectrophotometer. The molar
extinction coefficient used for insulin was 6200 M–1cm–1.13 (link)
in yeast (≥98% purity,
MW 5.8 kDa), protamine sulfate salt from salmon (MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR)
(MW 5.1 kDa), 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS),
and zinc sulfate monohydrate (ZnSO4) were purchased from
Sigma-Aldrich. Na2HPO4, NaH2PO4, NaCl, and phenol were purchased from Merck Chemicals Ltd.
All experiments were performed in phosphate buffer at pH 8.0 containing
10 mM Na2HPO4, 10 mM NaH2PO4, and NaCl. The concentration of insulin was measured using an absorbance
of 276 nm on the Cary 100 UV–vis spectrophotometer. The molar
extinction coefficient used for insulin was 6200 M–1cm–1.13 (link)
6
Characterization of Insulin-Protamine Complex
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Insulin human recombinant, expressed in yeast ([?]98% purity, MW 5.8 kDa ), protamine sulfate salt from salmon (MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR) ( MW 5.1 kDa), 8 anilino 1 naphthalenesulfonic acid ammonium salt (ANS) and zinc sulfate monohydrate (ZnSO 4 ) were purchased from Sigma Aldrich. Na 2 HPO 4 , NaH 2 PO 4 , NaCl and phenol were purchased from Merck-chemicals Ltd. All experiments were performed in phosphate buffer at pH 8.0, containing 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , and 100 mM NaCl. The concentration of insulin was measured using absorbance a 276 nm on the Cary-100 UV-Vis spectrophotometer. The molar extinction coefficient used for insulin was 6200 M -1 cm -19 .
7
Dual-Setting Hydroxyapatite Composites
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As inorganic reference, pure HA samples with a powder-to-liquid ratio (PLR) of 3 g·mL−1 were produced by mixing 2.5% disodium phosphate solution (Na2HPO4; Merck) with α-TCP raw powder. The obtained setting reaction sets to a matrix of calcium deficient hydroxyapatite (Ca9(PO4)5HPO4OH; CDHA) within several days. This process is shown in Scheme 3 .
For dual setting composites, the radical starter ammonium persulfate (APS; 0.5 wt%; Sigma Aldrich) was homogeneously mixed with α-TCP raw powder (99.5 wt%) using an electric coffee grinder for 60 s. Base of the liquid phase was a 2.5% Na2HPO4-solution (Merck) with addition of the radical catalyst N,N,N’,N’-tetramethylethylenediamine (TEMED; 2.5 wt%, Sigma Aldrich). Three different formulations were tested: (i) the first batch contained 40 wt% hydroxyethyl methacrylate (HEMA; H40; Sigma Aldrich) added to the liquid phase. A (ii) second formulation combined the short-chain monomer HEMA (40 wt%) with the synthesized polymeric cross-linker PEG-PLLA-DMA (10 wt%; H40P10). Third mixture (iii) was based on the pure synthesized PEG-hydrogel precursor (25 wt%; P25). The selected PLR was 3 g·ml−1 for all three formulations. Cement setting to HA as well as radical polymerization was initiated by homogenous mixing of cement powder and liquid on a glass slab.
For dual setting composites, the radical starter ammonium persulfate (APS; 0.5 wt%; Sigma Aldrich) was homogeneously mixed with α-TCP raw powder (99.5 wt%) using an electric coffee grinder for 60 s. Base of the liquid phase was a 2.5% Na2HPO4-solution (Merck) with addition of the radical catalyst N,N,N’,N’-tetramethylethylenediamine (TEMED; 2.5 wt%, Sigma Aldrich). Three different formulations were tested: (i) the first batch contained 40 wt% hydroxyethyl methacrylate (HEMA; H40; Sigma Aldrich) added to the liquid phase. A (ii) second formulation combined the short-chain monomer HEMA (40 wt%) with the synthesized polymeric cross-linker PEG-PLLA-DMA (10 wt%; H40P10). Third mixture (iii) was based on the pure synthesized PEG-hydrogel precursor (25 wt%; P25). The selected PLR was 3 g·ml−1 for all three formulations. Cement setting to HA as well as radical polymerization was initiated by homogenous mixing of cement powder and liquid on a glass slab.
8
Protein Extraction from Cell and Nerve Samples
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SCs from all experimental groups of the in vitro model, plated in 6 well-culture dishes, were scraped in 200 μL of RIPA lysis buffer [500 mL stock solution: 1.6 mM NaH2PO4 (Merck®), 8.4 mM Na2HPO4 (Merck®), 0.1% TritonX-100 (VWR®), 0.1 M NaCl (Ambion®), 0.1% sodium dodecyl sulfate (SDS; Fisher Scientific®) and ddH2O] supplemented with sodium deoxycholate (Merck®), 1 mM sodium fluoride and 1X protease and phosphatase inhibitor cocktails (Roche®). Protein concentration was quantified using a Bicinchoninic acid (BCA) protein assay (Bio-Rad®) in a SpectraMax microplate reader (bioNova científica®).
Using a Swann-Morton® scalpel, frozen sciatic nerve segments (5 mm) were chopped on dry ice and resuspended in 150 L of RIPA lysis buffer [500 mL stock solution: 1.6 mM NaH2PO4 (Merck), 8.4 mM Na2HPO4 (Merck®), 0.1% Triton X-100 (VWR®), 0.1 M NaCl (Ambion®), 0.1% sodium dodecyl sulfate (SDS; Fisher Scientific®) and ddH2O] supplemented with sodium deoxycholate (Merck®), 1 mM sodium fluoride with both 1X protease and 1X phosphatase inhibitor cocktails (Roche®). Samples were then homogenized in a Precellys 24 tissue grinder (Bertin Technologies®).
Using a Swann-Morton® scalpel, frozen sciatic nerve segments (5 mm) were chopped on dry ice and resuspended in 150 L of RIPA lysis buffer [500 mL stock solution: 1.6 mM NaH2PO4 (Merck), 8.4 mM Na2HPO4 (Merck®), 0.1% Triton X-100 (VWR®), 0.1 M NaCl (Ambion®), 0.1% sodium dodecyl sulfate (SDS; Fisher Scientific®) and ddH2O] supplemented with sodium deoxycholate (Merck®), 1 mM sodium fluoride with both 1X protease and 1X phosphatase inhibitor cocktails (Roche®). Samples were then homogenized in a Precellys 24 tissue grinder (Bertin Technologies®).
9
Synthesis and Purification of Organic Compounds
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LC and LF were procured from Sigma. CMF was prepared by the method of Fukumachi and Sakurai.22 (link) KA and DQ were prepared from RF by the methods of Surrey and Nachod50 (link) and Miles et al.,51 (link) respectively. The side-chain of KA and DQ was cleaved by periodic acid51 (link) oxidation. The compounds were purified by cellulose column chromatography using 1-butanol–1 propanol–acetic acid–water (50 : 30 : 2 : 18, v/v) as solvent system.18 (link) All the reagents and solvents of the highest purity were procured from Merck & Co. The buffers used were citric acid–Na2HPO4 (pH 2.5–8.0), H3BO3–KCl–NaOH (pH 9.0–10.0) and Na2HPO4–NaOH (pH 11.0–12.0). The ionic strength was 0.001 M in each case.
10
Electrochemical Experiments with Aqueous Solutions
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H2SO4, HCl, NaNO3 (Chempur), KCl (Sigma-Aldrich), NaOH (Fluka), and HCOOH (Sigma-Aldrich) were obtained. NaH2PO4 (Sigma) and Na2HPO4 (POCh) were used for aqueous electrolyte preparation with deionized water purified by using an Elix system (Millipore). Argon N5.0 was from Multax.
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