Hello, I have been using Chemidoc XRS+ for Western Blotting the last three months (I am new at this), and sometimes I have got a strange issue. After doing all the steps and placing the PVDF membrane into the Chemidoc, the camera does not reveal anything, just as if nothing had been placed inside the device (picture attached). I make use of the next protocol: After electrophoretic separation of my sample proteins, I tranfer them to a PVDF membrane by using Trans-Blot® Turbo™ Transfer System. Then, I block it for 1 hour at room temperature with PBS-Tween 0,5% and incubate it overnight at 4°C with the primary antibody. Next I do is wash 5 times with PBS-Tween 0,05% (10 minutes each time), incubate for 2 hours with the secondary antibody at room temperature, and wash 5 time again with PBS-Tween 0,05%. Finally, I pour the Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer) and, after 3-5 minutes, I drain it and reveal the membrane with the ChemiDoc system. Does anyone know about this? I have been working with this protoco for the last month, and I hadn't had problems until now. Thank you very much.
The first thing to do is to make sure the instrument is working properly. There should be some kind of luminescence standard available to test this, or see if someone else in the lab is using it successfully for ECL.
First of all, thank you for the answer. My workmates use the same ECL Plus kit that I use, and it works fine, so I think that is not the problem.
If you are using a new primary antibody that has not been used before, that could be the problem. You may have to increase the concentration by a large factor.
The next things I would check are the secondary antibody and the ECL reagent. Try using the ones that other people are using to get good blots. Yours may have gone off.
I used the primary antibody the last week and it went well. The ECL reagent is the same the other people are using. I will try to use another secondary antibody. Thank you
I have tried another secondary antibody and the same has happened. It is very strange, because nor even the protein marker bands show up in the picture. On the other hand, we usually mark the protein marker bands with a permanent black pen, not directly on the membrane, but on the plastic we use to cover it. We do it because frequently these bands are hardly visible in the picture, and the permanent black pen marks can be seen easily. The point is, regarding my issue, these black pen marks do not even appear.
Any suggestion, please?
I have tried to pour the ECL reagent over the other side of the membrane, just in case the proteins had been transfered there somehow. I obtained the image attached.
It is like if something in the membrane Surface was preventing the ECL reagent from working.
Any idea?
We all use the same type of membrane in our laboratory, so it should not be the problem.
The charging means, did you use methanol to dip the membrane in it before starting the blotting procedure. if not then you need to do this.
We use the Trans-Blot® Turbo™ Transfer System, which brings PVDF membranes that are used directly without previous dipping or any kind of preparation.
Thank you for the answer anyway.
Raúl Jiménez Tizón
I have the same problem as you show in picture one. Could you find out by now what the problem was? :)
Which equipment did you use? And I had the same problem versadoc system. I think it was about veradoc, but I have not found any solution
The first thing I would check, when you don't have anything on your blot, is if the transfer was performed succesfully. Do you have a control (like a prestained ladder) in order to determine the transfer onto the PVDF membrane?
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