Western blot: Circular bands - artefact? - (Jun/04/2007 )
Hi everybody!
Recently I did two Western blots against Actin for use as an internal loading control. The protein samples are derived from human skeletal muscle cells. I did the same experiment several weeks ago and everything was just fine.
Now the problem is that the actin does not show up as a regular band but rather as kind of a black circle or square around a white area. I repeated the blot and got similar result again. Those circular bands are completely different from the effects that you see when you had air bubbles in the blotting apperature. The effect can already be seen in ponceau staining before antibody detection.
I would be very grateful if anybody would have an idea how this artefact could develop!
Greetings, Timm
Photos of Ponceau S staining and westernblot detection (2 exposures) are attached.
Recently I did two Western blots against Actin for use as an internal loading control. The protein samples are derived from human skeletal muscle cells. I did the same experiment several weeks ago and everything was just fine.
Now the problem is that the actin does not show up as a regular band but rather as kind of a black circle or square around a white area. I repeated the blot and got similar result again. Those circular bands are completely different from the effects that you see when you had air bubbles in the blotting apperature. The effect can already be seen in ponceau staining before antibody detection.
I would be very grateful if anybody would have an idea how this artefact could develop!
Greetings, Timm
Photos of Ponceau S staining and westernblot detection (2 exposures) are attached.
too much antigen or primary Ab; reduce the amount of protein per slot; as muscle is a rich source for actin, you have a very high specific portion of actin in your samples...
The Bearer is right. The white places are where your HRP reaction has run out.
thanks a lot!
i will strip the membrane and try it again this week.
But if I've got it right, this effect can already be seen prior to antibody detection, so it should have nothing to do with antibody or antigen concentrations. To me it looks like two distinct bands running very close to each other giving only the impression of squares. This could mean you have two different isoforms of the protein. Maybe your previous gels with only one single band were run for a shorter time than the recent ones so the two forms were not separated.
ellis