The revelation of nitrocellulose membranes by chemiluminescence (ECL kit) showed white bands on a dark gray background for all protein extracts. These bands darkened gradually with increasing exposure time in the apparatus. I would ask if anyone has encountered this situation or propose any suggestions to resolve this problem.
I am on vacation till the 6 th of January and i don't have any pictures yet. The background of the picture is getting darker and the profile of the proteins with ponceau staining are exactly the same after revelation. For some wells were we loaded pure proteins and we see spots in this same proportions loaded (sometimes where we should see nothing). The antibody used is the Anti carboxymethyl lysine CML
I’ve repeated this experiment and I still have the same problem. In other similar experiments we used the anti AGE antibody (advanced glycation end products) and it works except for only one in which we had the same problem with the CML. Dilutions: Ac anti-CML : dilution : 1/1000 anti HPR 1/1000 (as recommended in notice) and 1:5000; blockage : PBS-tween(1mL/1L)-Gelatin (2.5g/L).
We'd have to see the picture, but I suspect Paul Lesbats is right. Every time I've seen this happening is because the amount of protein at that spot saturates the binding capacity of the membrane. Then, during the initial antibody binding step, protein molecules loosely bound at the upper 'layers' of that spot bind the detecting antibody, only to be washed off during subsequent steps. The net result is that they block the membrane even better than your blocking agent, making the blot look like a negative.
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