FAQ
Can you strip and re-probe ECL Plex probed blots?
Yes, we have detailed information in the ECL Plex protocol of a compatible stripping solution containing ß-mercaptoethanol. Usually, you do not need to strip and re-probe ECL Plex blots, since you can do multiplexing directly.
I have had problems when I scan my ECL Plex blots. I sometimes see areas that are much lighter and have weaker signals. Why is that?
Most likely, this is caused by partial drying of the membrane. The areas that have dried give very different intensities compared to the ones that are still wet. The membrane can start to dry on the scan bed. We recommend to always dry the membranes before scanning for best results (lower background and better signal to noise ratio).
How do I know that the membrane is low fluorescent? And, can I use the same membrane for 2D western?
How do I know that the membrane is low fluorescent? And, can I use the same membrane for 2D Western Blotting?
We have 2 low fluorescent membranes that we recommend for ECL Plex. One NC, Hybond ECL and one PVDF alternative called Hybond LFP. You can try other membranes, but then you have to test them first. If they give elevated background, there is risk that you loose the weak signals. And yes, you can also use them for 2D Western Blotting.
I have used ECL Plex and I have always a lot of background in the Cy3 channel. So ECL Plex Cy3 does not work so well for me. What can be the problem?
For lowest background, we recommend to scan the membranes dry. Also the scan setting is important. If you use a Typhoon 8600, the Cy3 filter setting will automatically be 555 BP 20, which is suboptimal and give high background and weak signals. Make sure to use the 580 BP 30 Cy3 TAMRA filter setting for optimal Cy3 detection.