实验原理 Western Blot采用的是SDS-PAGE,被检测物是蛋白质,“探针”是抗体,“显色”用标记的二抗。经过PAGE分离的蛋白质样品,转移到固相载体(例如硝酸纤维素薄膜)上,固相载体以非共价键形式吸附蛋白质,且能保持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋白质或多肽作为抗原,与对应的抗体起免疫反应,再与酶或同位素标记的第二抗体起反应,经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。该技术也广泛应用于检测蛋白水平的表达。 实验步骤 1. Resolve sample proteins and controls via polyacrylamide gel electrophoresis. Transfer proteins to nitrocellulose using standard methods.
2. Remove the blot from the transfer apparatus and soak in TTBS (Tween-Tris Buffered Saline: 0.1% Tween-20 in 100 mM Tris-CL [pH 7.5], 0.9% NaCl) for two rinses of 15 min. each. The blot may be marked (with pencil or India Ink) for identification at this stage if desired.
3. Block the blot with 10% nonfat dried milk (NFDM) freshly made in TTBS; rock on a rotating shaker for 15 min. at room temperature or overnight at 4°C.
4. Rinse the blot 3 times in TTBS.
5. Probe with primary antibody in TTBS/1% NFDM for 1 hr. at room temperature. Primary antibody should be diluted as specified on product data sheets. If these values are not available, use the following guidelines for initial experiments: apply antisera or ascites at 1:500 to 1:5,000, apply purified primary antibodies at a concentration of 1 ug/ml.
6. Rinse the blot three times in TTBS.
7. Probe the blot with an enzyme-linked secondary antibody (typically horseradish peroxidase or alkaline phosphatase) in TTBS/1% NFDM for 30 min. at room temperature. Review instructions included with the secondary antibody to determine the appropriate dilution to use.
8. Rinse excess secondary antibody from the blot with 3 rinses in 20-50 ml TTBS for 5 min. each. The blot is now ready for use with standard colorimetric or chemiluminescent detection reagents.