which is more sensitive: IP or western blot? - Looking for transcription factor - would IP help? (Aug/27/2007 )
I have a transcription factor that should be expressing under plant stress, but I haven't been able to find it using western blot. I am using Invitrogen Western Breeze kit.
I load ~ 1mg of protein (this is quantitated using a NanoDrop 280 reading) and then dilute my primary 1:1000 and 2ndary 1:1000.
My control is e coli made his-tagged transcription factor and it always shows up.
I was thinking of doing an IP first using my antibody because I would be concentrating my transcription factor then using the elution for my western blot.
I say I can't find my protein by western - that's after about a 4-5 minute exposure. Some of you may say its there and I just need to reduce my background so I can leave my film on longer, 20 - 30 minutes?
Thanks - H
a lot of transcription factors are indeed expressed at very low levels, but using 1 mg of protein seems a lot. Do you load all this in 1 single normal lane?
The IP is more sensitive than Western, as you can indeed perform the IP first if your antibody is suitable for this.
Putting on the film is something I would definitely do, some ECL reagents are active for a long time, while others are only active for a couple of minutes, so it depends on the one you use. However, we routinely put films on the blot for more than 15 minutes.
I have left my film on for 5 hours or more without having high background and can pick up bands that were not visible after a 5 min exposure. I use Invitrogen x-cell sure lock system, NuPAGE gels and PVDF membrane. I block and hybridize with 5% nonfat milk in 1x TBST and wash in 1x TBST for at least 30 mins with several wash changes. I visualize with Pierce Supersignal ECL.
After film exposure, you may wash the membrane with 1xTBST three times.
Reblot the membrane with secondary antibodies and finally ECL.
This may amplify the signal 2-3 fold
Hope this may help.
I would try an IP, but couple the beads to the antibody to maximize yields.
Are you sure you are breaking the nucleus? Because some buffer are more gentle and give only the cytoplasmic fraction... So maybe your method is right, but you don't see your protein because your cells are not completely "broken".