Impact factors on results of semi-quantitative western blot in pharmacological research / 中国药学杂志

ABSTRACT

OBJECTIVE: To investigate the impact factors of the semi-quantitative Western blot (WB), offering a guidance on research of pharmacological effects of drugs at the level of proteins. METHODS: Total proteins were extracted from the Huh7.5 cells infected with hepatitis C virus and quantified with BCA kit, HCV Core protein was chose as target protein, and protein Tubulin or Gap-dh, which was encoded by housekeeping gene, as the internal control. By controlling the experimental factors, such as loading sample amount, concentration and the incubation time of first antibodies, and dilutions of chemiluminescent fluid as well, we analyzed those factors how to impact the semi-quantitative results. RESULTS: The semi-quantitative results showed that there is a linear relationship between relative intensity of target protein and the amount of total protein at must protein concentration range, beyond which, the range-ability of relative intensity of target protein reduced, even no changes. However, sample volume loaded, or protein selected as internal reference has little influence on the result of semi-quantitative WB. In the context of obtaining high-quality band, concentration and incubation time of antibodies or dilution of chemiluminescent fluid also has no influence on it. Yet, the semi-quantitative WB has certain defects and our results show that the permissible error of semi-quantitative result should be controlled within 15%. CONCLUSION: The key impact factor on the result of semi-quantitative WB is the target protein amount of loading. On the premise of obtaining clear and high-quality bands, appropriately selecting loading volume of samples, concentration and incubation time of first antibodies, and dilutions of chemiluminescent fluid is conducive to accomplish experiments without affecting the final results of semi-quantitative WB.