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PostPosted: Wed Sep 04, 2013 7:56 pm 
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Joined: Wed Sep 04, 2013 7:41 pm
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Good day everyone! I have come hoping that someone may have some helpful advice to help me with my westerns that are being, well, tempestuous to say the least. I am relatively new tot he technique as I am more of an analytical chemist by training who has transitioned into a biochem lab. As I have been learning the technique I have been using prepared lysates from Santa Cruz Biotech. I have been utilizing Nupage 4-12% bis-tris gels and my first couple westerns where I probed for b-actin with an MCF7 lysate worked well, but then I started to get transfer issues. We have been using the iBlot system from Invitrogen (Life Technologies) and have always used program 3 for 7 minutes.

So this is what I typically do:
1. Load my samples in a titer of 25 ug- 2.5 ug of protein, load PS and SS markers
2. Run gel in 1x MOPS buffer
3. Transfer for invitrogen PVDF stack and run program 3 for 8 minutes (lately I have tried out increasing the time up to 12 minutes to no avail)
4. After transfer I coomassie stain my gel and I Ponceau my membrane. I find that I MAYBE get justa few bands that show with the Ponceau and the coomassie after destaining shows LOTS of protein remaining

I have tried using program 0 and increasing the run time of program 3, nothing has helped. After working with Life Tech tech support they suggested I get a new iblot. Same thing, it wasn't my iblot.

Any help would be much appreciated. Thanks!!

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