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What species of bone cells were being tested? Murine Osteoblasts from calvaria What dilution of primary antibody for each? OPG 1:100; ATF4 1:400; RANKL 1:500 Was any other antibody used successfully with these same samples? No How much protein was loaded on the gel? 20 ug Please, could you tell us when you'll ship us the antibodies in substitution?
Asked on Nov 16 2011
Thank you for providing the additional information. The one thing I would like to recommend in this case is to run a "loading control" western blot with the murine osteoblast cell lysate. Examples of targets for this type of control would be proteins that are commonly expressed at high levels in all cell types, such as actin or GAPDH. This would serve as a control in that detection of the expected band size would indicate that all aspects of the lysate preparation and western blot protocol are working as expected. Thanks so much for your troubleshooting efforts! I look forward to hearing how things go.
Answered on Nov 16 2011