Detecting phosphorylated PKR - antibody and positive control - (Sep/17/2006 )
Hello,
I am going to detect phosphorylated PKR in cultured cells. It seems that Western blotting with anti-phospho PKR antibody is the easiest way of doing this. Has anyone of you had such experience? Which companies sell the working antibody including Abcam, Cell signaling, Upstate, etc. and which positive control to use (poly I:poly C, IFN-alpha, gamma, etc.)
Thank in advance you for your help.
We had tried to detect PKR also but failed. I dont remember the company which supplied it.
We needed a positive control and we couldnt induce it with only PolyI:C, or IFN- gamma.
We switched to detecting IFN-alpha with a kit. But we needed to induce the cells with LPS and IFN-gamma and detected some IFN-alpha.
Good Luck !!!
Thank you Malik!
This experiment is very important for me. People have used polyI:C, or IFN+calyculin A, to induce phopho PKR. Companies which sell anti-phospho antibody also show Western result with cells such treated. I hope either will work.
This experiment is very important for me. People have used polyI:C, or IFN+calyculin A, to induce phopho PKR. Companies which sell anti-phospho antibody also show Western result with cells such treated. I hope either will work.
Also some one suggested to us that some cell types might not react to these compounds bcoz of lack of certain receptors etc.. So take time to look at the literature more closely.
Good Luck !!!
I just want you to know that my experiment turned out as expected. I purchased antibody for PKR and phospho-PKR from Cell Signaling and treated my cells with 1000 U interferon alpha for 24 hrs and then with 0.1 uM calyculin A for 15 min. The treatment induced phospho-PKR very nicely as detected by Western blotting. Thanks for helping.