big black spot in the place of my protein on western... - (Feb/10/2006 )

hello again....I did western and all I can see is black spot i guess in the place of my protein.....maybe i didnt wash enough sec antibody? i can see some other bands but cant specify them because of the big black spot.... so im washing again....but dont know if thats going to work...what should I do? what could be a problem?

IMHO, a big black spot on a blot (it always occurs where your band of interest is) is a result of incomplete blocking.

Was your blot completely immersed in blocking solution?
Was there no air bubbles of any appreciable size in contact with your blot?

If you can answer yes to these two questions, then it may be the cause of your problem.


I block and do my antibody application in a hybridization bag and am paranoid about removing all air bubbles. I think it makes a difference.

I also believe this is the problem of blocking process.

If you are using non-fat milk for blcoking, please make sure milk powders are completely dissolved before use.

thanx everybody we firgured out the problem....the X-ray film was exposed to light and so....was destroyed..... ....but anyway the bands were very very intense...so i think i have loaded too much protein and also blocking was bad.. but what was strange is that band came out in the same place as positive control...(no GST) and my sample has GST>...should the band be in the different place? couldnt see the marker also....

thanx everybody we firgured out the problem....the X-ray film was exposed to light and so....was destroyed..... ....but anyway the bands were very very intense...so i think i have loaded too much protein and also blocking was bad.. but what was strange is that band came out in the same place as positive control...(no GST) and my sample has GST>...should the band be in the different place? couldnt see the marker also....