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Mysteriously disappeared bands

Mysteriously disappeared bands - Western Blot Forum

Mysteriously disappeared bands - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 02-02-2013, 12:03 PM
Pipette Filler
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Exclamation Mysteriously disappeared bands



Hi all! i am new here

i have read about bands that gone missing, so here is my very own mystery:

Repeating a WB experiment using the same recipes,tested antibodies etc as before, i reached the final step of developing the image out of my membrane. Using ECL for 5 min and then incubating another 5 min the membrane with the film into the cassette,I developed the image and it was ok; i saw my bands but there was background so i put my membrane into PBS-Tween (the recipe I always use) to wash it for about 30 min (5 min-washes in a total time of 30 min) in order to get a clearer image. The thing is, when I tried to develop the image again (ECL: 5 min, cassete: 5 min), all bands were lost, not a single sign. I changed ECL (truth is mine was old), but still not a sign of my bands, the film was clear. I changed films, fresh NEW (tested) ECL, I varied the time period of incubating the membrane with the film in the cassette (giving it more time), still not a thing (I gave it more than 10 shots). Finally, I incubate my membrane with aTubulin (tested antobodies first and secondary) and I tried to develop it (using my old ECL for 5 min and then incubate the membrane with the film for 2 min. Normally, atubulin bands appear HUGE after this period). I saw 3 very weak bands (though my samples were 6) so i incubated again the membrane with the film for 10 min expecting HUGE signal this time. When i developed it,... NOTHING, not a sign, not even the 3 weak bands.

So, is it possible that my membrane somehow "died out" during the PBS-T wash because after the washes I never saw my bands again ? I suspected the old ECL,too, that's why I used a fresh new one, but still there was no sign of my protein bands. And which is, after all, the best PBS:Tween? (I usually use e.g. 200ul of Tween in 200ml PBS but i have been told a great variety)

any ideas/have u experienced the same mystery?? sure thing, I am going to repeat the WB...
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