Protein lobind

Xylan (1% w/V) was hydrolysed with 1 µg/ml of xylanase in the presence or absence of (i) EnzHR lignin from steam pretreated spruce (1% w/V) or (ii) a buffer soluble fraction of EnzHR lignin in a concentration corresponding to the concentration to buffer soluble lignin in experiment (i). Background controls used were xylan, lignin and enzyme with lignin. Hydrolysis was performed in triplicate in 50 mM Na-citrate buffer, pH 5 in 40 or 50 °C for up to 48 h with mixing in 200 µl volume in protein low binding tubes (Protein LoBind, Eppendorf). Reducing sugars were analysed using the PAHBAH assay [59 (link)] after terminating the reaction by incubation at 98 °C for 10 min. The effect of monomeric phenolic compounds on the hydrolysis of xylan was analysed in similar conditions as described above, but instead of lignin, a phenolic compound, protocatechuic acid, syringic acid, ferulic acid, vanillic acid, p-coumaric acid, acetovanillone, vanillin, syringaldehyde or homovanillyl alcohol, was added to the reaction in 10, 100 or 1000 µg/ml concentration and a protein low binding 96-well plate (Protein LoBind, Eppendorf) was used.

After aspirating the media, the cells were treated with TrypLE™ Express, incubated, and rinsed with pre-cooled 1X PBS. The content was collected into individual sterile Eppendorf Protein LoBind^® microtubes and centrifuged (500× g, 4 °C; 10 min). The produced supernatant was discarded, but the soft, transparent pellet was collected and lysed with ice-cold lysis buffer (200 μL of RIPA, protease inhibitor 20% v/v, phosphatase inhibitor 1% v/v) and incubated (4 °C; 20 min). Following that, the cell suspension was homogenized using an ultrasonic cell crusher and then briefly centrifuged (2000× g, 4 °C; 10 min). The proteins extracted were collected into new, individual, sterile Eppendorf Protein LoBind^® microtubes and were concentrated using a speed-vacuum concentrator (300 rpm; 24 h; 60 °C) before storage at −152 °C for subsequent analysis.

Aliquots of 10 µL streptavidin-coated magnetic beads were added to an Eppendorf Protein Lobind 500 µL 96-well plate and washed twice with 200 µL 0.1% TWEEN20 in 1 × PBS. In this and all following washing and incubation steps, the magnetic beads were isolated by letting the plate stand for 5 min on a 96-well magnet plate from Alpaqua Magnum FLX (Beverly, MA, USA) and removing the wash solution using a BenchTop Pipettor from Sorenson Bioscience Inc. (Murray, UT, USA). Next, aliquots of 100 µL freshly diluted biotinylated capture antibody solution (4.00 µg/mL) in 0.1% TWEEN20 in 1 × PBS were added. The samples were incubated at 37 °C and 900 rpm for 120 min using an Eppendorf Thermomixer® Comfort to allow binding of the biotinylated capture antibody to the streptavidin-coated magnetic beads. Next, the magnetic beads were isolated and washed twice with 200 µL 0.1% TWEEN20 in 1 × PBS. Aliquots of 100 µL of plasma or serum were then added and incubated at 37 °C and 900 rpm for 120 min to allow binding of human growth hormone to the bead-capture antibody complex. Next, the magnetic beads were isolated and washed twice with 300 µL 0.1% TWEEN20 in 1 × PBS and twice with 1 × PBS. After overnight storage at + 4 °C, GH was eluted off the beads with 25 µL 0.1 M citric acid solution in water:acetonitrile (90:10, v/v) for 10 min at 45 °C and 900 rpm.