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We are testing whole tissue homogenate from pig kidney, using proteanase inhibitor and phosphatase inhibitor. The sample was also sonicated. We used anti-angio-1 (ab8451) antibody first, and the results showed in the second picture. Then we switched toanti-angio-1 (ab95230)antibody, the results showed in the first picture. 5% dry milk in TBS w/ 0.1% Tween-20 was used to block and dilute the antibodies. The primary antibodies were diluted 1: 1000 and incubated over night at 4 degrees. The secondary antibodies were diluted 1: 10000 and incubated at room temperature for 1 hour.
Asked on Jan 04 2012
Thanks for providing the western blot images as well as the protocol and sample preparation details. I just have a few additional questions: 1. Was any other antibody usedsuccessfully in western blotting with these same samples? 2. How much protein was loaded per lane? 3. Angiopoietin may be glycosylated and may contain disulfide bonds. This may account for bands at higher molecular weights than expected. Was reducing agent used in sample prep? 4. Are the samples in each lane of the both blots the same, ie. is the lane 1 sample in the ab8451 blot the same sample as that run in lane 1 on the ab95230 blot? Thanks in advance for the additional information.
Answered on Jan 04 2012