I have done a Southern Blot Analysis of DNAmt transferred to nylon membranes. The DNA was firstly loaded on a 2% agarose gel. An immunoassay was done to detect the bands using Anti-Digoxigenin antibody with Alkaline Phosphatase which catalyzes a colouring reaction with NBT and BCIP.

The results has a very strong background as you can see in the following pic:

enter image description here

I am not sure about what can be the reason of that background. I have read that thick agarose gels produce strong backgrounds, but I never experienced that before. If somebody has experience on this or alternative explanations, I would thank him/her to share them with me.

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    $\begingroup$ Could you please describe your procedure? It is extremely difficult to pick apart what may be wrong here when we have no idea how you made the gel, how long you placed the anti-body on, what you blocked with, etc etc. 2% is not a terribly thick gel. It's higher than the norm (~1%) but I've never had this trouble with them. My guess is you did something weird when placing the antibody or blocking. Perhaps made the buffer improperly. $\endgroup$ – FrankyG Dec 21 '15 at 21:59
  • $\begingroup$ I agree, try to post the full protocol step-by-step (e.g. 1, 2, 3) since sensitivity can be altered by a number of mishaps that can occur at different steps of the process! $\endgroup$ – CKM Dec 21 '15 at 23:21
  • $\begingroup$ Thanks FrankyG and Kendall. I will write a short traslated version of the protocol and will link it here in a while. It is the first time I follow it but I know it worked fine for other colleagues. Probably I did something wrong and am trying to figure out what. When I tell a thick gel I mean the mm of thicknes. I have read that "The recommended thickness for agarose gel is 3–4 mm; a gel thicker than 5mm will result in fuzzy bands and higher staining background." Source: thermofisher.com/us/en/home/life-science/pcr/… $\endgroup$ – Oscar Moya Dec 22 '15 at 8:25
  • $\begingroup$ The protocol bit.ly/1QE3y7z $\endgroup$ – Oscar Moya Dec 22 '15 at 9:53
  • $\begingroup$ Offhand, I'd look at the blocking and washing steps (perhaps try a different source of nonfat dry milk). Be careful about contamination (alkaline phosphatase is ubiquitous). Also, you may be over-exposing - reducing reaction time might produce cleaner results. At the extreme end, you might try using peroxidase-linked rather than phophatase-linked antibodies. $\endgroup$ – R.M. Jan 13 '16 at 19:05

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