Tris hcl

After the incubation with the studied extracts at 100 μg/mL, the supernatants above the cell cultures were collected. 20 μL of the supernatants were loaded on a 10% polyacrylamide gel with addition of 0.1% gelatin (POCH) and 0.001% SDS (Sigma). Electrophoresis was performed at 120 V (starting at 90 V, until the face reached the separating gel). Afterwards, the gels were washed twice (2 × 15 min) in renaturing buffer (2.5% Triton X-100 (Sigma) in 50 mM Tris-HCl (Sigma) followed by two 15-min washes in 50 mM Tris-HCl buffer. Subsequently, the gels were incubated in incubation buffer (5 mM CaCl₂ in 50 mM Tris-HCl) at 37 °C for 24 h. After the incubation, the gels were stained with 0.15% Coomassie Brilliant Blue R-250 dissolved in methanol: acetic acid: glycerol: water (16:2:1:23, by vol.) for 30 min. and destained in methanol: acetic acid: glycerol: water (8:2:1:29, by vol.)^{37 (link)–39 (link)}. Densitometric analysis was performed using Image Studio Lite software (LI-COR Biosciences).

The Hexokinase assay was performed as previously described^{87 (link)}. Briefly, 20 µg of fresh tissue lysate was added to 1 ml of reaction buffer for hexokinase (50 mM Tris HCl (Sigma-Aldrich), pH 7.5, 10 mM MgCl₂ (Sigma-Aldrich), 0.6 mM ATP (Sigma-Aldrich), 100 mM glucose (Sigma-Aldrich), 0.2 mM NADP+ (Sigma-Aldrich), and 0.1 units of glucose-6-phosphate dehydrogenase (Sigma-Aldrich)). Ten units of glyceraldehyde-3-phosphate dehydrogenase (Sigma-Aldrich) per ml was used for analyzing the Hexokinase activity. The Pyruvate kinase assay was performed as previously described^{87 (link)}. Briefly, 20 µg of fresh tissue lysate was added to 1 ml of reaction buffer for pyruvate kinase (50 mM Tris HCl (Sigma-Aldrich), pH 7.5, 5 mM MgCl₂ (Sigma-Aldrich), 5 mM ATP (Sigma-Aldrich), 0.2 mM NADH (Sigma-Aldrich) 100 mM KCl (Sigma-Aldrich), 5 mM Na₂HPO₄ (Sigma-Aldrich), 5 mM MgCl₂ (Sigma-Aldrich), 0.01 mM AMP (Sigma-Aldrich)) 5 mM fructose-6-phosphate (Sigma-Aldrich), 5 units of triosephosphate isomerase (Sigma-Aldrich) per ml, 1 unit of aldolase (Sigma-Aldrich) per ml was added to check the pyruvate kinase activity. A negative and positive control has been included without tissue lysate and with 0.05 units of hexokinase and pyruvate kinase in both the assays. Enzyme activities were measured and represented as the change in absorbance/min, calculated using a linear portion of the obtained curve.

Five different lysis buffers, listed in Table 1, were used to lyse the cell pellets. The supernatant was analyzed by SDS PAGE. The buffer with highest yield of soluble recombinant protein was selected for further studies. From each buffer 2 mL were added to the cell pellet from a 5 mL IPTG-induced culture and sonicated for 5 min duration. The resulting lysed sample was centrifuged at 13,000 rpm for 10 min and the supernatant was then purified.Table 1

Buffers and their composition used in the optimization of expression of recombinant SLSP-k.

S. no.	Buffer	Final concentration
1	Tris–HCl (Merck), pH 7.5	20 mM
	Dithiothreitol (DTT) (Sigma-Aldrich)	0.1 mM
	Lysozyme (Sigma-Aldrich)	1 mg/mL
2	Tris–HCl (Merck), pH 7.5	20 mM
	NaCl	0.5 mM
	Lysozyme (Sigma-Aldrich)	1 mg/mL
3	Phosphate buffer	20 mM
	NaCl	0.5 mM
	Urea	8 M
	Triton-X 100	1%
4	Phosphate buffer	20 mM
	NaCl	0.5 mM
	Triton-X 100	1%
5	Phosphate buffer	20 mM
	Triton-X 100	0.5 mM

Bovine serum albumin (BSA) and Coomassie Brilliant Blue G-250 were purchased from Sigma-Aldrich Co. Ferric chloride hexahydrate (FeCl₃ · 6H₂O), ferrous chloride tetrahydrate (FeCl₂ · 4H₂O), 3-(triethoxysilyl)-propylamin (APTES), ethanol (96%), tetraethoxysilane (TEOS), trichlorotriazine (TCT), and thetrahydrofuran (THF) were prepared from Merck. NaH₂PO₄ · 2H₂O, Na₂HPO₄ · 12H₂O, and tris-HCl were purchased from Merck and used for preparation of phosphate and tris-HCl buffers at pH = 7.5.

The chromatography experiments were performed at room temperature of 20°C using an ÄKTA avant 25 (Cytiva, Uppsala, Sweden). A device of 0.4 mL of volume, consisting of a cellulose-based fiber matrix with geometry and flow properties similar to the commercially available HiTrap Fibro™ PrismA was provided by Cytiva (Uppsala, Sweden) (Hardick et al., 2013 (link); Dods et al., 2015 (link); Cytiva, 2020b ). The fiber matrix is functionalized with an affinity ligand designed for purifying AAVs serotypes, including 1, 2, 3, and 5, similar to the commercially available affinity chromatography resins Capto AVB or Sepharose AVB (Cytiva, 2018 ; Cytiva, 2020a ). The device was equilibrated with 15 column volumes (CV) of 20 mM Tris-HCl (Merck KGaA, Darmstadt, Germany), 500 mM NaCl (Merck KGaA, Darmstadt, Germany), and pH 8. After sample loading, the unit was washed with 30 CV of equilibrium buffer. The AAVs were eluted with 40 CV of 100 mM Glycine (Sigma-Aldrich, Missouri, United States), pH 2.5 and neutralized with 1 M Tris-HCl (Merck KGaA, Darmstadt, Germany), pH 9.

LC–MS grade solvents were purchased from VWR Hungary (Debrecen, Hungary). Ammonium formate, ammonium acetate, formic acid, Tris-HCl, Ca(OH)₂, glycerol, and chondroitinase ABC were from Sigma (Budapest, Hungary). Heparan sulfate and chondroitin sulfate disaccharide standards and heparin lyase I-II-III enzymes were purchased from Iduron Ltd. (Cheshire, UK).