Author Topic: problems with ECL Prime  (Read 15447 times)

LauraM

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problems with ECL Prime
« on: August 13, 2012, 07:10:01 AM »
We have huge problems with westernblotting since we have to use ECL Prime instead of ECL Plus.
We didn't change our protocol except for the primairy antibody dilution (2x less concentrated) and the developing time (shorter)
We use ECL hyperfilms.

What is the problem? we don't get signal or we lose the signal.
For example. I take my blot, incubate it 5 min on RT with 1:1 ECL Prime. I drain off excess ECL and place my blot between plastic sheets.
Then I expose my hyperfilm for let's say 5 min. I develop it and see signal/bands but it is to weak. So ik expose again for let's say 15 min. But now
I don't see anything! The ECL seems to fade within 15-20 min every time. With ECL plus we could expose for more than a hour and still see signal.

But the strange thing is I got a good result just once. I had my film with nice bands, but it had lots of background so I washed it in PBS shortly and without new ECL I exposed again. It was perfect. Then I had to do something else en about an hour later I exposed for a second time and still I got a great signal. So the ECL was still emitting light after more than a hour and after washing with PBS.
Due to irregular loading I repeated this westernblot in exactly the same way (protocol, Ab's and proteins) and now I can't get a signal when I expose for a second time (15-30 min) after the ECL. So the first shortly exposed film gives bands and the longer exposed films after that give nothing. They are empty.

We have been trying different things (longer ECL incubation, the blots wet or dry, washing with PBS after ECL or not, different antibody's, second time ECL, second time Ab incubation, longer washing steps, etc.) for 3 weeks now with 3 different people and we can't get the ECL to last longer than 15-30 min.  A second problem is that if the first exposure was a succes and we do a second incubation with a loading marker (b-actin) we don't see actin. So the end conclusion is that we see bands the first exposure and all the exposures that we do after that fail no matter what we try.

So we are desprate by now, because we can't get nice films due to the ECL that fades very fast, and we don't know why. So can you help us? Maybe you can give some suggestions for what the problem might be ans what we can try to fix it?

Susanne GE

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Re: problems with ECL Prime
« Reply #1 on: August 21, 2012, 07:58:47 AM »
Hi, sorry to hear about your problems. I have been working  with the development of ECL Prime and have quite much experience from it. I have done a lot of studies to look at the stability of the signals in comparison with for example ECL Plus. My experience and data shows very cleraly that Prime is more stable than ECL Plus. However, this may not help you. I am pretty shure that your problems are related to the antibody dilutions. ECL Prime has another chemical composition than ECL Plus an also much higher light output. Therefore ECL prime require lower concentrations of both primary and secondary Ab to obtain a good balance in the reaction. The quickly fading signal you obtain is probaby caused by substrate depletion. It is important to know that ECL prime is not identical with ECL plus and may require other conditions. Best is to optimize the antibody dilutions.
You say that you have increased dilution of primary Ab but you probably need to dilute both primary and secondary Ab further. It is difficult for me to give you exact recommendations when I do not know your protein and Ab dilutions. However, I can give you some general guidelines, here it comes.
Dilute the primary Ab 2-4 times more compare to ECL Plus, in general sometines between 1:3000-1:5000, depending on Ab (sometimes you need to go even higher, up to 1:7000)
Dilute the secondary Ab also  2-4 times more compare to ECL Plus, generally a dilution of 1:50000 works but may be increased to 1:70000.
Wash 6x5 min after each Ab incubation.
Add ECL Prime, incubate for 5 min, drain off and expose. Sometimes it gets even better if you wait 5-10 min before exposure.
Another possibility for you could be to also decrease the sample amount a bit. In your case I definitely think the major issue is that you have to high concentartions of secondary Ab. Please try my recommendations. I hope this will help. If you still having problems with the signal stability and/or high background, please contact your sales rep, we may be able to help you further. you can even contact me directly at: susanne.grimsby@ge.com

Kind regards
Susanne
 

LauraM

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Re: problems with ECL Prime
« Reply #2 on: August 22, 2012, 03:37:33 AM »
Hi Susanne,

Before you're reply I searched on the internet for possible causes. I found a ECL Prime video on YouTube and after watching that I decided to do
what you just suggested. I used a primairy Ab. diluent of 1:2000 instead of 1:500 and a secundairy Ab dilution of 1:20.000 instead of 1:1000. I was
a little bit "scared" to dilute it further. But this time I had a signal, it was very strong and I only had to expose for a few seconds and it didn't fade.
So yes, the problem is the Ab dilution. So the next step is to dilute even further so that the exposure time get's a bit longer.
So thank you for you're answer! at least i'm now sure that this is or was the problem.

Greetings Laura