Anti gfp rabbit polyclonal antibody

Recombinant pGM-CSF or BHK21 cells transfected with VenusC1-pGM-CSF were suspended in 1×Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA). Constituent proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) then transferred onto PVDF membranes (Millipore) for WB as previously described (32 (link)). Membranes were blocked with 1% BSA in PBS and probed with our home-made antibodies against pGM-CSF (mAb-2A4H11 or rabbit polyclonal Abs) and anti-GFP rabbit polyclonal antibody (Proteintech, Wuhan, China). A home-made PRRSV-N specific mAb-6D10 and commercial tubulin mAb (Transgene) were used for detection of corresponding target. Specific binding of antibodies to corresponding targets was detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (Transgene) and visualized using ECL substrate (Bio-Rad Laboratories). Chemiluminescence signals were recorded digitally using a ChemiDoc™ MP system (Bio-Rad Laboratories). Digital signal acquisition and densitometry analyses were conducted with the ImageLab Program, Version 5.1 (Bio-Rad Laboratories).

Cells were grown in YPD medium at 30 °C overnight and diluted to an OD₆₀₀ of 0.1, in YPD medium containing 150 μM BPS to deplete iron. Cultured cells were harvested, diluted to an OD₆₀₀ of 0.1 in YPD medium containing 150 μM BPS with or without 100 μM FeCl₃, and incubated at 30 °C for 12 h. Cells were harvested by centrifugation and resuspended in a protein extraction buffer comprised of 50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycolate, 1 mM PMSF, and a protease inhibitor cocktail (Sigma, Munich, BY, Germany). Cell lysates were prepared by bead-beating, and the protein concentration was determined by the Bradford assay [17 (link)]. Total proteins were separated on a sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma, Munich, BY, Germany). Protein detection was performed using an anti-GFP rabbit polyclonal antibody (Proteintech, Rosemont, IL, USA) or an anti-FLAG (Abcam, Cambridge, UK) rabbit polyclonal antibody. A mouse anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz, Dallas, TX, USA) was used as a secondary antibody, followed by visualization by chemiluminescence.