Weak and variable signal blotting for MDR1 using C219 monoclonal Ab - (Apr/19/2007 )
I've been using the C219 mouse monoclonal to probe for MDR1 expression in epithelial cells (T84 and Caco-2). I only sometimes get a signal and it can be very variable from one blot to the next.
I've tended to use it at 1:50 and blocking and Ab dilutions are done with 3-5% nonfat dried milk in TBS-Tween. I've tried blotting at RT for 2hrs and overnight at 4 degrees. I use a HRP conjugated secondary at 1:5000 for 1hr. I use Pierce Pico chemiluminescence substrate for detection.
My blots vary between no signal or diffuse smears. Has anyone used this Ab before or do you have any suggestions that may help?
Does it vary from lot to lot of the antibody, or are you using the same tube for all experiments? It could be that your gel or transfer is different or incomplete from time to time.
I've used two seperate lot numbers with similar effect. I spoke to the company rep who informed me they had no reports of any problems with either lot number.
It's a membrane protein and large (170kD). I'm wondering whether this is part of the problem, perhaps with isolating the hydrophobic protein. I've also tried different transfer conditions - overnight vs 2 hours, varying methanol and SDS % etc. The problem is getting clean results and I'm simply not sure whether it isn't just a poor antibody!
Thanks for your response WAstate