I have noted that when I reveal some PVDF membrane using ECL, different parts of membrane show different intensity of signal. (i.e. stronger signal on borders than in the middle).
Among possible reasons you might want to consider:
1) The volume of your 1st and/or 2nd antibody is not enough to cover all the area of your PVDF membrane. As a result, during the incubation on the rotation platform (especially, at high speed of rotation) the edges (borders) of your membrane have more exposure to antibody solution, and, therefore, show higher intensity of the signal.
2) During the ECL development of your membrane the mixed ECL solutions did not cover the middle of the membrane, because of the air bubble(s) trapped between the glass surface and the plastic wrap your membrane is located on. Therefore, the signal is more intense in the areas (edges) where the enzyme has more ECL substrate available to generate a stronger signal.
Among possible reasons you might want to consider:
1) The volume of your 1st and/or 2nd antibody is not enough to cover all the area of your PVDF membrane. As a result, during the incubation on the rotation platform (especially, at high speed of rotation) the edges (borders) of your membrane have more exposure to antibody solution, and, therefore, show higher intensity of the signal.
2) During the ECL development of your membrane the mixed ECL solutions did not cover the middle of the membrane, because of the air bubble(s) trapped between the glass surface and the plastic wrap your membrane is located on. Therefore, the signal is more intense in the areas (edges) where the enzyme has more ECL substrate available to generate a stronger signal.
I have also noticed that this can happen if you seal the baf for the incubation with the primary to near to the edges of the blot. ntibosy than does not reach the sides.
Ihope that helps
Thanks Viktor and Elena. I am gonna check the shakers here. Actually they shake on a weird direction (I have noted this as well). Hope my problem will be solved. Best,.
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