How to get rid of the background? - My blot (May/16/2008 )
How can I get rid of the background? It is getting more intense everytime I am doing the blot. How frustrated! Sometimes it is even worse that it is actually black or dark in colour at the side of the blots.
I had tried to use TBS only to wash the blot before developing the film. I put 1ml of developing reagent per blot with pipette and wait for 1 minute (I suspect the problem is here).
I washed plenty of times for a long time and still getting background. Is it better to wash more after primary antibody or secondary antibody?
Thanks guys, attached file is the sample of the blot. This is more presentable than my others (cause I didnt scan them yet, too embarrassing to use them).
Cheers and have a nice weekend!
If nothing else works, use Erase-It Background Eliminator Kit (pierce product). It works great in our lab, although personally I have never required it.
that background looks like it comes from a dirty dish and/or processed without agitation or insufficient volume.
since you say it is getting worse, i would look at the processing dish and see if it is being thoroughly cleaned (sometimes residue, that you may not be able to see, builds up on the plate).
we had this kind of problem with our silver stains. we started cleaning the boxes with sodium thiosulfate to clean the residue.
we've also seen this kind of background on our westerns and had to clean the processing vessel with alcohol and elbow grease (we use colorimetric detection). background cleaned up.
mdfenko, when you mean processing dish, does that mean the containers used for washing etc? Hmmm I will try to clean that up before using. I usually just rinse it with water and use it.
I suspect the electroblot problem but I aint that sure. I have to change the electroblot and find out about it. thanks! Any more suggestion?
I can't purchase the background kit. My supervisor will kill me!
We had blots like this as well. In our case it was due to old film developing solution in the dark room. We prepared it fresh and voila the background was gone...
It is anyways not good to depend upon such kits. I would also suggest borrwoing different company films to see if some films work better. This is of course after you have reasonably eliminated all other possible causes for background.
not just the washing but also the vessel that you use to incubate with the other solutions. if they are reused then they may be contaminated.