Anti rabbit igg horseradish peroxidase conjugate

Reagents were purchased from the following sources: anti-mouse IgG horseradish peroxidase conjugate, middle range molecular weight protein markers, Promega; anti-rabbit IgG horseradish peroxidase conjugate, anti-bovine IgG horseradish peroxidase conjugate, anti-bovine IgG alkaline phosphatase conjugate, anti-equine IgG alkaline phosphatase conjugate, diaminobenzidine (DAB), fibrous DEAE-cellulose, benzamidine, iodoacetamide, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), leupeptin, phenyl methyl sulfonyl fluoride (PMSF), Sigma; prestained high molecular weight protein markers, Gibco BRL; 5-bromo-4-chloro-3 indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), protein assay dye reagent concentrate, bovine serum albumin (BSA), broad range molecular weight standards, and nitrocellulose membranes (0,45 μm pore size), BioRad. All other chemicals were of the highest quality grade available.

Cells (0.1 × 10⁷) were cultured in a 6-well plate (Nunc) and incubated with and without rfhSP-D (20 µg/ml) in a serum-free DMEM-F12 for various time points. The cells were then mixed with 2× treatment buffer (50 mM Tris–HCL pH 6.8, 2% v/v β-merceptoethanol, 2% v/v SDS, 0.1% w/v bromophenol blue, and 10% v/v glycerol) and sonicated for 30 s before running on a SDS-PAGE (12% w/v) for 90 min at 120 V. The SDS-PAGE separated proteins were electrophoretically transferred onto a nitrocellulose membrane using an iBlot 7-min Blotting System (Thermo Fisher), followed by blocking with 5% w/v non-fat dried milk powder (Sigma) in 100 ml PBS for 2 h on a rotatory shaker at room temperature. The membrane was washed with PBST (PBS + 0.05% Tween 20) three times, each time for 10 min. The membrane was then incubated with primary anti-human TGF-β antibody, (1:1,000; R&D systems) at 4°C overnight on a rotatory shaker, followed by secondary anti-rabbit IgG horseradish peroxidase-conjugate (1:1,000; Promega) for 1 h at room temperature. The positive bands were visualized using 3,3′-diaminobenzidine (DAB) substrate kit (Thermo Fisher).