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