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Trouble with actin, HA and CCND1 :(( - (Jul/09/2010 )

I've been doing western for ages, but getting no results!!

We extract proteins in the lab from our cell lines and estimate them in NanoDrop.
I'm checking to see if there's over expression of cyclin D1 (tagged with HA) in my clones. However, the trouble is, the HA antibody doesn't seem to be working. I've tried it 5 times to no avail. I then tried antibodies against cyclin D1 and it worked! but only once B)
So, i tried actin, and it worked too! also only once :) (the second time i got some black patches on my film, maybe non specific binding)
I can't seem to be getting any reproducible results.
can anyone suggest what the problem might be?


A few details about what blotting /probing system you are using would be good... I am guessing 5% milk powder in PBS or PBS-T?