Dear all,
we are currently having trouble with our Westernblots.
Attached you can find two pictures of our blots.
We use fluorescently labelled antibodies, but even if we do not incubate the blots after transfer with the ABs, we see this problem. So it seems so be some kind of autofluorecence.
Changing the buffers, using different water and so on, nothing really help. Sometimes, the blots look ok and sometimes these red clouds occur, without any relation...
Does anybody have an idea what to do or change to get rid of this?
Thanks in advance.
Maren
I think that you didn't do sufficient washing of your membrane. or while doing transfer, bubbles must be present which were not squeeze out at the time of transferring the protein from gel to membrane.
The dots come from particulates that are stuck to the membrane. This could be from the blocking agent if it is not completely dissolved. Also, if you are using a low percentage gel, bits of the gel may become stuck to the membrane and cause the dots.
The clouds may be due to insufficient blocking, or, if you are using chemiluminescent detection, from the luminescent chemical swirling around in an excess of liquid surrounding the blot.
These sorts of problems are more severe when the detection is weak, resulting in long imaging times that bring up the background.
The red clouds e.g. are even present, if we strip the blot completely and scan again or don´t even incubate the membrane with antibodies at all....
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