Recombinant hg csf

Transgenic and non-transformed Wolffia plants (in an amount of 1 g) were grinded in liquid nitrogen and resuspended in 3x volume extraction buffer (50 mM Tris-HCl (pH7.6), 150 mM NaCl, 5 mM EDTA, 5 mM β-merkaptoethanol, 0.62 μM aprotinin, 8.4 μM leupeptin). Extraction was performed during 40 min at 4°C followed by centrifugation for 20 min at 16,000 g; supernatant was collected for subsequent analysis. Protein concentration in the samples was determined by Bradford method (Bradford, 1976 (link)).
Protein electrophoresis (70 μg/lane) was carried out in 10–25% gradient SDS-PAGE. The separated proteins were transfered onto a nitrocellulose membrane (BioRad, USA). Rabbit polyclonal antibody against hG-CSF (diluted 1:1,000; Abcam, UK) and anti-rabbit IgG conjugated with alkaline phosphatase (1:3,000; Pierce, USA) were used. Recombinant hG-CSF (Abcam, UK) served as the positive control. The blots were visualized using chromogenic substrate BCIP/NBT (Fermentas, Lithuania).

Chlorella cultivated in N-deficient media were harvested by centrifugation at 8,000 rpm for 20 min, and then, the spent medium and Chlorella were sampled. For extraction of total protein from Chlorella cells, 50 mg of cells frozen in liquid N were homogenized in a mixer mill MM300 (Qiagen, Germany) and then resuspended in 1 M PBS buffer (pH 7.4) including 1 × cOmplete™ (Roche, Germany) at 4 °C. The solution was centrifuged at 14,000 rpm for 10 min, and then, the supernatant was transferred into a new tube. To determine the concentration of total proteins in the medium, the spent medium was filtered through a 0.2 μm membrane and was concentrated up to 250 times via 5 kDa size cut-off Viva Flow 200 and Vivaspin 20 in turn (Sartorius, USA). After the cell lysates and total proteins from the medium were separated in 12% NuPAGE gel (Invitrogen, USA), they were transferred onto nitrocellulose membranes using the TurboTransfer system (Bio-Rad, USA). After the membrane was incubated with the polyclonal antibody of hG-CSF (Abcam, USA) for 1 h (1:2000 dilution), it was exposed to anti-rabbit IgG-HRP, which was diluted to 1:5,000 (Abcam, USA). Recombinant hG-CSF synthesized in CHO cells (Abcam, USA) was used as the positive control.

The total protein was extracted as described above. The protein samples were serially diluted in PBS and loaded into 96-well ELISA plate (0.25, 0.5, 1.0, and 2.0 μg of TSP per well), using recombinant hG-CSF (Abcam, UK) as the reference standard.
The plates were incubated for 2 h at RT, then washed three times with PBST (PBS with 0.1% Tween 20) for 5 min each and blocked in PBST containing 2% bovine serum albumin (1 h at RT). Hybridization with the primary antibody was performed overnight at 4°C. After washing, the secondary antibody was added and plates were incubated for 1 h at RT followed by the washing. Rabbit polyclonal antibody against hG-CSF (Abcam, UK) was diluted at 1:1,000, anti-rabbit IgG conjugated with alkaline phosphatase (Pierce, USA)—at 1:2,000. The plates were developed for 30 min at RT using TMB Peroxidase EIA substrate (BioRad). The plates were read at 405 nm and the amount of plant—expressed hG-CSF was estimated based on reference standards.