Biosep sec s4000

Size-exclusion high performance liquid chromatography (SE-HPLC) measurement was carried on a Shimadzu LC-20AT HPLC system equipped with a RF-20A UV-vis Detector (Tokyo, Japan) according to the method of Chaudhary et al. [16 (link)]. Gluten proteins with different proportions of KGM, added for different frozen periods, were extracted by adding 50 mg of the freeze-dried gluten protein samples to 1 mL of an acetic acid solution (500 mM). The insoluble part that accounted for about 20% of the total gluten protein samples was removed by centrifugation for 10 min at 5000 r/min. The soluble part of gluten protein sample was filtered through a 0.22 μm PVDF membrane filter and then subjected to SE-HPLC analysis. A 20 μL gluten protein sample was injected into a size-exclusion column (Biosep-SEC-S4000, Phenomenex, 300 × 7.8 mm, Torrance, CA, USA). The samples were eluted with an acetic acid solution (500 mM) at a flow rate of 0.8 mL/min and were detected at 280 nm. A calibration curve with r² > 0.9 was obtained by plotting the peak area of the ribonuclease (Mw 1.37 × 10⁵ Da), ovalbumin (Mw 4.43 × 10⁵ Da), γ-globulin (Mw 1.50 × 10⁵ Da), and bovine thyroglobulin (Mw 6.70 × 10⁵ Da), all of which were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Chaetomium thermophilum communities were enriched from cell lysates by spin filtration and fractionated using a Biosep SEC‐S4000 (7.8 × 600) size exclusion chromatography (SEC) column from Phenomenex, Germany (see Appendix Supplementary Methods).

Size‐exclusion HPLC was performed using a Phenomenex™ BioSep‐SEC‐S 4000, 300 × 7.8 mm, 5 µm column (No. 313834‐4, Phenomenex; Torreance, CA). The mobile phase was PBS, and the flow rate was 0.75 mL/min for 25 minutes. The ChemStation analog‐to‐digital converter (Agilent Technologies; Santa Clara, CA) was set to 25 000 units/mV, peak width 2 seconds, slit 4 nmol/L. [¹²⁵I] was detected with a raytest Ramona 90 (raytest USA, Inc; Wilmington, NC) in line with a standard Agilent 1100 HPLC module system. The data were collected using ChemStation for LC 3D Systems (Revision B.01.03 [204], Agilent Technologies).

The protein solubility was assessed by SE-HPLC
Waters 2690 separations module and a Waters 996 photodiode array detector
(Waters, U.S.A.) using a Biosep-SEC-S4000 (300 Å ∼ 4.5
mm) with a prefilter SecurityGuard GFC 4000 (Phenomenex, U.S.A.) using
a three-step extraction procedure, all at room termperature, described
fully in Gällstedt et al.^{11 (link)} It will
be described only briefly here. In the first step, 16 mg of each foam
sample was suspended in 1.4 mL of 0.5 wt % SDS buffer (pH 6), and
the supernatant (SN) was obtained after a centrifugation at 2000 rpm,
followed by 5 min agitation. The second extraction (Ext. 2) was performed
by resuspending the residual sample after the first extraction in
a new SDS buffer solution and sonicating it in a ultrasonic disintegrator
for 30 s. The third extract (Ext. 3) was obtained by resuspending
the residual sample after the second extraction in a new SDS buffer
solution and sonicating it over a longer period of time (30 + 60 +
60 s). Three replicates per formulation were used. The SE-HPLC analysis
was performed, using a 0.2 mL/min of an isocractic flow consisting
of 50% acetonitirile, 50% Millipore water, and 0.1% trifluoracetic
acid.

Aβ oligomers were prepared as described above. The size of prepared Aβ peptides was analyzed by SEC. Samples were run on a BioSep SEC-S 4000 (Phenomenex) column using a ProStar 210 HPLC (Varian) system with a ProStar 325 UV detector (Varian). Injected samples were eluted with PBS (pH 7.4) at a flow rate of 0.5 ml/min at ambient temperature, and data were obtained at 280 nm. Peak areas were integrated using Star 6.2 Chromatography Workstation (Varian).