Sephacryl s 300 column

Unless otherwise stated, all extraction procedures were performed at 4 °C. Fresh eggs were homogenized in a blender with 3 volumes (v/w) of 0.4 M NaCl for 30 min and then centrifuged at 4500 rpm for 10 min. The supernatant was then dialyzed for 24 h to obtain water-soluble glycopeptides. An equal volume of 90% phenol was added to the supernatant with mild stirring overnight to destroy the protein 3D structure while retaining glycoprotein stability. After centrifugation, the aqueous phase was dialyzed and lyophilized and the crude samples were then applied to a QFF column (GE Healthcare, USA) using the AKTA™ UPC 100 (GE Healthcare) system eluted by a linear gradient (0–1 M) of NaCl in 0.02 M Tris-HCl buffer (pH 8.0). The elution profile was monitored by measuring the absorbance at 230 nm for proteins. The carbohydrate profile was assayed using the phenol–sulfuric acid method. As shown in Figure 1A, the Q-4 fraction, sialic acid-rich fraction, was further chromatographed using a Sephacryl S-300 column (GE Healthcare) and eluted with ultrapure water. The S-3 fraction containing the most protein, carbohydrates and sialic acid was freeze-dried and used as the experimental sample.

Recombinant His-GBA3-HA and GST-NEU2 proteins were prepared in the presence of 1× complete protease inhibitor cocktail (Roche Diagnostics K.K.) and 1 mM Pefabloc (Roche Diagnostics K.K.). Aliquots of the two proteins were incubated together overnight at 4 °C. A 0.5 mL aliquot of each protein or mixture (1.5 mg/mL of each) was loaded on a Sephacryl S-300 column (GE Healthcare Japan; 1.5 × 50 cm) equilibrated with elution buffer (300 mM NaCl in 50 mM Tris-HCl, pH 7.5), respectively, and fractions of 0.9 mL were collected. Fractions were assayed for enzyme activity, and 0.8 mL of each fraction was precipitated with trichloroacetic acid (10%) and further subjected to SDS/PAGE and western blotting.

SAXS data of hIL-12, in the absence and presence of heparin, was acquired at the Cornell High Energy Synchrotron Source (CHESS) beamline G1 source. Inline size exclusion chromatography (SEC) was used to minimize polydispersity. hIL-12 (150 µM), with and without low molecular weight heparin (in ten-fold excess) was loaded onto a Sephacryl-S 300 column attached to an AKTA explorer (GE Healthcare). Eluted protein sample entered the flow cell of the BioSAXS at CHESS and was subjected to X-ray beam exposure. Beamline characteristics used for acquiring the data were as follows: energy = 9.968 keV(1.257 A); beam diameter = 250 µm × 250 µm; photon flux = 1.6 × 10¹¹ photons/sec. The detector used was a dual Pilatus 100K-S with a q-space range between 0.006 to 0.8 Å-1. Data acquired was processed using BioXTAS RAW software for performing the buffer subtraction. Buffer subtracted plots were analyzed using the ATSAS program^{35 (link)} with a sequence of steps to obtain an average low resolution structure.

Myosin was extracted from rabbit skeletal muscle and purified using the technique of Margossian and Lowey 24. Subfragment‐1 (or S1) was produced by chymotryptic digest of synthetic myosin filaments 25. The S1‐bearing supernatant was dialyzed against 50 mm imidazole pH 7.0, 0.3 mm EGTA, and 1 mm DTT and cleared by centrifugation. This supernatant was then adjusted to 150 mm NaCl and purified by size exclusion chromatography on a Sephacryl S‐300 column (GE Healthcare, Piscataway, NJ, USA). The purity and composition of full‐length myosin and the S1 subfragment proteins were confirmed by SDS/PAGE 26.

rHDL was prepared from purified rh-apoA-I using the Jonas sodium cholate dialysis method with a molar ratio of apoA-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine:cholesterol of 1:100:10 (79 (link)). rHDL particles were purified by gel filtration chromatography with the use of a Sephacryl S300 column (GE Healthcare) on a Bio-Rad Biologics DuoFlo FPLC (Bio-Rad), as previously described (11 (link)). The size of rHDL containing apoA-I was estimated from 4 to 20% nondenaturing equilibrium gel electrophoresis (using precast gels; Bio-Rad) by comparison with protein standards of known Stokes diameter (GE Healthcare) as previously described (77 (link)). Nondenaturing gels were then analyzed by Coomassie blue staining. The amount of cholesterol was determined using a LiquiColor enzymatic cholesterol test kit (Stanbio Laboratory). Total phospholipid content in the rHDL was quantified by microphosphorous assay (80 (link)).