Rabbit polyclonal antibody against 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).

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.