HELP:protein over-expression failure?! - (Jul/02/2008 )

I inserted a 3.5kb new gene into pcDNA3.1(-) ,with a flag tag at the N terminal. Then transfected it into HEK-293A cells. Collect cell protein after 48&72 hours. But western blot using anti-flag antibody showed no expected band. What possible problem there might be?

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Eh, 934 reasons! Some here:

1. Western did not work. Controls?

2. Transfection did not work. Controls?

3. Cloning of 3.5 Kb was bad. Out of frame, promoter messed up, polyA gone etc. Sequencing? RE digests?

4. Bad Flag Ab. Controls?

I hope somebody will take it up from here..

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