terrible streaks on western bands - (May/11/2006 )
hi! i'm working on a 55kda protein runx2 in MC3T3-E1 cells (mouse osteoblast). I use nuclear extract for this. I happen to detect the western bands with some other proteins but I'm really having a hard time with this protein. I can't seem to get a clear band, there's always a presence of dots and streaks. I stain my membrane after transfer with ponceau-s and bands appear perfectly well. But when I incubate it with the primary and secondary antibodies, the bands look so terrible. see the attached image.
i usually boil my samples for 2 mins at 90C since i noticed that prolonged boiling could make my bands worse. I use PMSF, DTT and aprotinin in my extraction buffer. should i add more protease inhibitor?
i hope somebody could help me. I'm really starting to become desperate. 
Buy a new packet of skim milk powder.
Agreed - it's presumably the blot milk.
And make sure the suspension is homogenous...
thanks for the immediate response...but i'm just using the same blotting milk with other proteins. how come it only happens with Runx2 (transcription factor)?
Buy a new packet of skim milk powder.
Agreed - it's presumably the blot milk.
And make sure the suspension is homogenous...
would it help if i decrease the blotting milk from 5% to 3% for antibody incubation??
I encountered a similar problem before. Your antiserum seemed to have reacted with human keratin. Keratin bands run approximately 55-60 kDa. You may get rid of them by wearing gloves, washing glass plates extensively and preparing new electrophoresis buffer. Do not touch buffer, glass plates and other stuff with your bare hands. Hope it helps.
wow! that made sense...coz sometimes i could get clear bands with the same protein. i will try to be cautious next time!!! hope to get perfect bands next time!
thanks a lot! 
MC3T3 have low endogenous Runx2 level. You could try more protein per line. Not all antibodies good for Runx2 in these cells, best we tested are Mouse monoclonal to C-terminal part.