Chemilluminscence issues - (Jul/29/2005 )

few questions:

do you apply the chemilluminescent solution in the dark room?

how long do you expose your membrane??

my westerns are just not working and im getting worried...does anyone have any suggestions????

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make an overnight exposure and see how, sometimes your protein sample was just not enough to give you signal in several minites...

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i never heard its neccesary to put it on the membrane in the dark room.
as it goes for the exposure, it depends on your solution! usually ecl works from 20min to 1h, so there is no point exposing the film longer.
check stuff youre using - some solutions are more stable, so you can try longer.

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if you get very weak signal in your IB experiments try ECL Plus kit from Amersham...

i have also a question: is it necessary to preincubate ECL solutions from 4C to RT or 37C before mixing for increasing rate of the chemioluminescent reaction?

Seb_

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i usually bring them to RT before mixing. never tested if 4 / RT makes any difference, and never tried 37, so i dont really know. however HRP is an enzyme, so in theory temperature should have some effect (that's why the time of secondary ab incubation should not exceed 1h), i would guess at least for reproducibility.

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use an ecl with higher sensitivity. for example, pierrce fermo ecl.

good luck!

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Are you using a brand new or a really old ECL kit? It may be that the solution is bad. You could test it by spotting a microlitre or two of your secondary antibody on a piece of membrane then add the ECL reagent to that and see if it shows up. It should be glowing if the kit is good.

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Hi Sashrafi,

I usually expose from 30 secs up to 3 minutes. If my protein is not showing in this time, I expose it for up to 45 min.

Putting the ECL on the membrane in the dark room is not necessary, does no harm if you put in on under light. I prewarm it to room temperature, the earlier starts the reaction.

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