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i recently developed a b-actin-ab-incubated PVDF-WB-Membrane with the Thermo Scientific Pierce ECL Kit. According to the manufacterers orders i mixed Reagent 1 and Reagent 2 1:1. But unfortunately i got kind of "unbalanced" bands (on the right side of the picture). Then someone gave me the tip to try to mix the reagents 2:1. For this substrate i got the result on the left side which actually looks much better. Has anyone of you had the same experience and can anyone explain to me why this is so? Is it "legal" to do so, or does it alter somehow the results of my experiment?

Yours, p1ssn3lk3

WB with b-Actin-AB, developed with Thermo Scientific Pierce ECL

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    $\begingroup$ I have put some potential reasons for your observations, however it would be great if you expanded your question in terms of the the exact steps you followed. $\endgroup$ – Bez Aug 25 '14 at 10:15
  • $\begingroup$ Yeah you're right, i should add some more details :)! So it's a beta-actin-antibody which is already coupled with HRP, i didn't use a second antibody. I incubated on a roller board overnight in the fridge. I washed 3 times with TBS-T afterwards and also after the first ECL reaction i washed 3 times (10mins each) with TBS-T. $\endgroup$ – p1ssn3lk3 Aug 25 '14 at 11:04
  • $\begingroup$ when you did your second ECL, was it without any new Ab staining? $\endgroup$ – Bez Aug 25 '14 at 11:13
  • $\begingroup$ Thats it.. no new staining.. $\endgroup$ – p1ssn3lk3 Aug 25 '14 at 12:10
  • $\begingroup$ I think the problem is unequal ECL distribution and if you done immediate ECL second staining without washing the membrane thoroughly and waiting a while before second ECL. There is a chance that when you overlaid your film on the membrane, you didn't get a good contact. what is your exposure time? This can also be a one of results! to make sire of the difference, redo the experiment as I have discussed in the first paragraph of my response. p.s why do you use a single step staining? is that part of your project? $\endgroup$ – Bez Aug 25 '14 at 12:59
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Well usually in an ECL kit, one of the reagents is the substrate and the other one is hydrogen peroxide, which activates the substrate for breakdown using the HRP on your secondary antibody (Ab) so I genuinely doubt what you are seeing is the result of ratio difference. In order to make that conclusion you need to run your samples twice (on two separate membranes) and do a 1:1 and 2:1 ECL and if you observed such a difference then you are on firmer grounds.

I suspect what has happened in the above experiment is that you have done a 1:1 (with 1Ab and 2Ab not distributed equally on the membrane) then after perhaps not a very thorough wash, you have done another 1Ab and 2Ab incubation using the a 2:1 mix. I suspect what has happened is that in your 1:1 the left hand side has not received the Ab mixtures very well and when you did the 2:1 since the right hand still Ab attached to it (if you didn't strip the membrane) so the left hand side was "forced" to get the Abs hence again the Ab mixture and ECL not equally bound. It is obvious since one image is opposite the other so its the Ab incubation step that has gone wrong. Otherwise if you have done the second ECL incubation mixture straight after the first and didn't do a wash step, the same can result if the membrane did not receive the initial ECL mixture equally since you can get a blocking effect by the old ECL mixture and perhaps what has happened is that the H2O2 or the substrate was present in excess in the second incubation step hence amplifying the signal in the sections of the membrane that did not receive the ECL mixture very well in the first place, relative to the rest of the membrane, which still has ECL liquid on it from the last experiment (again if you haven't washed the membrane well as ECL can be active for up to 24 hours in some kits but after a few hours the activity usually reduces but can still be observed)

What you need to do is after ECL, wash the membrane with TBST for a few minutes followed by TBS, leave the membrane in the sun to dry so that the Ab slowly denature (although that doesn't always happen specially with high affinity Abs such as anti-tubulin and actin). After at least 24 hours, strip the membrane from Ab and redo the Ab incubation. The best thing to do is to do all the incubation steps in a sealed bag then make sure the membrane receives the ECL mixture equally and do the visualisation. I would say however since the membrane is unequally "contaminated" with the Abs then the best thing to do is to redo the experimental run and re-run the gel. Its a pain but you need to get to the button of this!

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  • $\begingroup$ U sun dry your blots? Really? $\endgroup$ – rhill45 Aug 25 '14 at 10:46
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    $\begingroup$ indeed! it helps reducing the signal from any previous Ab staining which uses the same secondary or even a different 2Ab sometimes, specially for anti-Tubulin! $\endgroup$ – Bez Aug 25 '14 at 11:09
  • $\begingroup$ Interesting stuff, I've always used BME but may try this sometime. $\endgroup$ – rhill45 Aug 25 '14 at 15:29
  • $\begingroup$ @buzrw after ECL wash with TBST first, then TBS and then just leave it on a filter paper to dry but then you need to go through methanol socking step etc once you need to re-use the membrane. If you found the response helpful would appreciate a vote. If you needed any further detail just leave a comment. $\endgroup$ – Bez Aug 25 '14 at 17:57
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I had a similar problem. In my case, I realized that it is absolutely essential to wash the membrane in TBS without Tween (TBS, not TBST) before you use ECL. If you have any amount of soap (Tween) on your membrane it reacts in some way with ECL and each time you should be getting a different pattern of these stains. Washing the membrane in TBS and then incubating with ECL made a difference for me and fixed the problem.

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    $\begingroup$ Pierce (or Thermo Fisher now) recommends washing the blots with a buffer containing Tween. It also doesn'z list Tween as a problem in the trouble-shooting section of the manual. And I haven't seen problems with detergents in my blots with ECL. It was rather a problem of incomplete mixing or distribution. $\endgroup$ – Chris Feb 11 '15 at 13:45
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Try rinsing the blot in TBS or PBS before treating. Shake it try and place on level dry surface. When I pour the a chemo reagent on there I just pour enough so the surface tension keeps the fluid on the paper not allowing it to run off.

Other possibilities I see could be in your transfer. The temp could not be uniform in the buffer

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