PDCD4 Plasmid, RAW 264.7 not working Help - (Sep/24/2012 )

I bought PDCD4 cDNA clone from Origne and amplified using Maxiprep system. I transfected the RAW 264.7 macrophage cell using Fugene reagent. Surprisingly, I found that all the PDCD4 level is gone as measured by western blot. I am really in trouble.

Can anyone help me? Has anyone faced such problem in RAW 264.7 macrophage?
Amit

Have you monitored your transfection efficiency?
Or you can transfect other easy-transfected cells(293T or other) for confirmating your constructs.
Good luck!

Transfection of macrophage cell lines are troublesome, especially because of the endosomal compartments ready to degrade delivered DNA in the cytoplasm. I have experience with J774A.1 cells. You need to carefully optimise the protocol... if applicable to your setup, you may also use an agent like chloroquine.

You may refer to: http://www.protocol-online.org/biology-forums-2/posts/23094.html

Good luck.

Gunes.