white bands on western blots - (Oct/26/2005 )
Hello, i am using a D2R antibody (santa cruz) and found that at the desired MW (49 and 51kDa) my bands are white and there are black bands at 75 and 150 kDa. my D2R ab is a goat polycolonal and my secondary is bovine-antigoat (i use 5% bovine serum to block or 3% BSA in TBS-tween) i tried diluting my antibody from 1:10k all the way out to 1:250k (and along the way also tried 1:50K, 1:100K, 1:200K) with little success. i also did a loading study and loaded as little as 2ug per lane-with no success. i have also tried diluting my primary from 1:1k all the way to 1:100k (and at the dilution with my secondary at 1:40k i can begin to make out bands- but this seems counter to what i have read that if you see white bands (or a reversed image) it is due to too much HRP-in the system- and even more puzzling- when i did a dilution of 1:50k with my primary and 1:20k with my secondary i say extremely white bands??? if anyone has encountered this problem before could you please give me some advice? anyone have any ideas? i would really, really appreciate it!
It has been mentioned before in this forum, something like "ghost band". You may have a search on these posts.