Hello,
Today I want to ask about ECL required for detection in western blot.
I prepare transparent vinyl to cover ecl-treated membrane units before
detection during western blot. I hope you will understand what I described as
'transparent vinyl' above because it is not sure for me to call it 'transparent vinyl'
After detection several times, I often wash membrane units and then treat
ECL additionally for further detections.
(ECL use differs depends on the situations, but
I never use ECL plus before general ECL.)
Currently I change transparent vinyl every time i began 'new detections after
quick washing' in particular experiment
but i can't remember whether I changed 'vinyls with ECL remained' in the
past when I conducted detections.
What I am concerned about is that ECL remained on transparent vinyl can affect the net
result of further detections with new ECL.
Actually, it seems that I have found no troubles in detections considering
my results, but it is just my subjective view and i think procedure should be taken into account more seriously than results.
I really hope I delivered what I wanted to ask to you well.
Thank you and good luck!
Any answers?
Hello dear Lee,
That was once my concern either. I believe, it isn't a problem according to my own experience (SuperSignal, Femto, Thermo). However, the brand (sensitivity) of the ECL might be another aspect of the case. You should take into consider this either.
Hope it was helpful,
Best regards
UA
Hello Ullvi Ahmadov,
Thank you so much for your informative reply!
So, you mean that mixing of ECL described in my qeustion does not affect the detection results?
I appreciate again.
I usually wipe the vinyl before usage everytime, mainly just to keep it clean and more durable. But whatever remained shouldn't cause much problem, since it would very little in volume even if still wet. When I have to switch ECL on the same blot, I would rinse the membrane and wipe the transparent vinyl just so that there won't be excessive amount of liquid in the cassette. So it's not really super clean. I would be concern about ECL effect if you see the blots having smudged background or dark wavy dark wavy pattern.
Dear Carrie K Lee,
I appreciate your answer with helpful information.
I learned a lot thanks to your help and great knowledge.
I want to make sure about several things : so you mean that you would concern about ECL effect after detection by your way?
And ECL remained in vinyl should not cause critical problems with detections in all cases? (Although nothing remained is the most optimal)
Thank you again.
ECL remained from previous usage should be minimal, I would suspect other issues first before looking into the cleanliness of the vinyl. For example, if you see dirty background, change the washing time, use different blocker. If the signal is weak or not detectable, look into the antibody.
Hello Lee,
Yes, I meant that.
You are most welcome.
Best regards and good luck!
UA
Hello Carrie K Lee,
really sorry for bothering you.
So you meant that even ECL remained on vinyl
caused by absence of wiping vinyl is minimal and does not
affect detection results critically?
sorry again
Thank you so much Carrie K Lee.
I am quite impressed with your great knowledge and patience for answering
my subsequent questions.
I got great help from you.
I appreciate again.
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