请教一个western的问题
丁香园论坛
1078
Heparin-Sepharose Precipitation and Immunoblotting
To analyze for CTGF protein expression, conditioned media were collected and the heparin-binding proteins were precipitated by end-over-end mixing for 4 h at 4°C with heparin-Sepharose CL-6B beads from Pharmacia (Piscataway, NJ). The beads were washed three times with ice-cold radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 2 mM ethylenediaminetetra-acetic acid). The bound proteins were then eluted by boiling in SDS sample buffer (62 mM Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol and bromphenol blue) for 5 min under either nonreducing or reducing conditions (containing 5% mercaptoethanol). The eluted heparin-binding proteins were resolved in 4 to 20% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose filters from Schleicher & Schuell (Keene, NH) for 2 h at 140 mA. The filters were blocked with blocking buffer (TTBS; 150 mM NaCl, 50 mM Tris, 0.2% Tween 20, 5% BSA, pH 7.4) for 2 h at room temperature and then probed for CTGF by incubation for 40 min with anti-CTGF antibody at 0.5 µg/ml in blocking buffer. After extensive washing at 37°C, the filters were incubated with either HRP-conjugated donkey anti-rabbit IgG from Amersham (Arlington Heights, IL) or HRP-conjugated rabbit antichicken IgG from Zymed Laboratories at a 1:12,000 dilution in blocking buffer. Immunoreactivity was detected by using the Super-Signal chemiluminescent substrate from Pierce (Rockford, IL), according to the manufacturer's instructions.
上面这种方被多数人用来作组织培养液和尿液的western ，我想用超滤管超滤离心尿液，将其浓缩并且提纯（而不用上面讲我看好象是在用肝素琼脂糖沉淀有点麻烦），然后上样再做western 可行吗？
帮帮忙吧！！！
To analyze for CTGF protein expression, conditioned media were collected and the heparin-binding proteins were precipitated by end-over-end mixing for 4 h at 4°C with heparin-Sepharose CL-6B beads from Pharmacia (Piscataway, NJ). The beads were washed three times with ice-cold radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 2 mM ethylenediaminetetra-acetic acid). The bound proteins were then eluted by boiling in SDS sample buffer (62 mM Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol and bromphenol blue) for 5 min under either nonreducing or reducing conditions (containing 5% mercaptoethanol). The eluted heparin-binding proteins were resolved in 4 to 20% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose filters from Schleicher & Schuell (Keene, NH) for 2 h at 140 mA. The filters were blocked with blocking buffer (TTBS; 150 mM NaCl, 50 mM Tris, 0.2% Tween 20, 5% BSA, pH 7.4) for 2 h at room temperature and then probed for CTGF by incubation for 40 min with anti-CTGF antibody at 0.5 µg/ml in blocking buffer. After extensive washing at 37°C, the filters were incubated with either HRP-conjugated donkey anti-rabbit IgG from Amersham (Arlington Heights, IL) or HRP-conjugated rabbit antichicken IgG from Zymed Laboratories at a 1:12,000 dilution in blocking buffer. Immunoreactivity was detected by using the Super-Signal chemiluminescent substrate from Pierce (Rockford, IL), according to the manufacturer's instructions.
上面这种方被多数人用来作组织培养液和尿液的western ，我想用超滤管超滤离心尿液，将其浓缩并且提纯（而不用上面讲我看好象是在用肝素琼脂糖沉淀有点麻烦），然后上样再做western 可行吗？
帮帮忙吧！！！
