Horseradish peroxidase hrp conjugated goat anti rabbit igg secondary antibody

Total proteins were extracted from the five cell lines (GES-1, NCI-N87, SGC7901, BGC823 and HGC-27), and protein concentration was determined by Bradford assay. Protein samples were then separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene fluoride membrane using a Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk powder at room temperature for 90 min, then incubated with rabbit anti-human KLF17 polyclonal primary antibody and rabbit anti-human GAPDH primary antibody as the loading control (1: 400 dilution; Abcam, Shanghai, China) overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Abcam) at 37°C for 1 h. After rinsing, the immunosignal was developed in a dark room and the membrane image was captured using a Labworks 4.6 system (Labworks, Novato, USA). The signal intensity (grey value) was then read with Image J software, and average absorbance of the protein bands was used to indicate the intensity of protein expression.

Western blotting was performed as described previously.^{13 (link)} Briefly, proteins were extracted from previously frozen cancer and
paracancer tissues by lysing them in radioimmunoprecipitation assay buffer
containing complete protease inhibitor cocktail (Pierce Chemical Company,
Rockford, IL, USA). The protein concentration was determined with the Bradford
assay (Bio-Rad, Hercules, CA, USA). Denatured protein (200 μg) was separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a
polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane
was blocked with 5% nonfat milk in Tris-buffered saline–Tween 20 for 2 hours,
then incubated with rabbit anti-vinculin (1:800; Abcam, Cambridge, MA, USA) and
rabbit anti-β-actin antibody (1:1000; Abcam) at room temperature for 12 hours.
The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG secondary antibody (1:2000; Abcam) for 2 hours after washing
with rinse buffer at room temperature. Membranes were developed with enhanced
chemiluminescence (Pierce Protein Research Products), and optical densities were
analyzed by ImageMasterTM2D Platinum (Version 5.0; Amersham Pharmacia Biotech
GE, Shanghai, China).