Author Topic: ECL Select instead of ECL Advance  (Read 8881 times)

Susanne GE

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ECL Select instead of ECL Advance
« on: March 30, 2012, 05:01:51 AM »
I have been using ECL Advance for detection in my Westerns because of the high sensitivity and the possibility to use higher dilutions of my antibodies although I sometimes get problem with to high background. I realized that ECL Advance is not available from GE Healthcare and I need to find another high sensitive reagent and saw that the newly launched ECL Select might be a good replacement.  What about the detection sensitivity and antibody dilutions for ECL Select, is it comparable to ECL Advance? Can I use the same conditions as for ECL Advance? Anyone who can advise me how to change from ECL Advance to ECL Select.

GE Maria

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Re: ECL Select instead of ECL Advance
« Reply #1 on: April 01, 2012, 11:51:41 PM »
Dear Susanne,

Amersham ECL Select has similar sensitivity as Amersham ECL Advance but requires higher antibody concentrations compare to ECL Advance. However, it is still possible to use high dilutions and ECL Select works with a wider range of Ab dilutions. I recommend using 3-5 times higher concentration for primary Ab  and 2-4 times higher concentration for secondary Ab compare to ECL Advance. A good starting point could be a dilution between 1:5000-1:10000 for primary Ab and 1:100000 for the secondary Ab. But as always remember that optimal Ab dilutions vary between different applications and depend on quality and affinity for the target protein. By using higher antibody concentration it is possible to reach even better sensitivity, stronger signal intensity and lower background than with ECL Advance.

Good luck!
GE Maria

wiger123

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Re: ECL Select instead of ECL Advance
« Reply #2 on: October 15, 2012, 09:44:47 PM »
I have been using ECL Advance for detection in my Westerns because of the high sensitivity & the chance to make use of higher dilutions of my antibodies although I sometimes get issue with to high background. I noticed that ECL Advance is not available from GE Healthcare & I need to find another high sensitive reagent & saw that the newly launched ECL Select might be a lovely replacement. What about the detection sensitivity & antibody dilutions for ECL Select, is it comparable to ECL Advance? Am I able to make use of the same conditions as for ECL Advance? Somebody who can advise me how to change from ECL Advance to ECL Select.

Susanne GE

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Re: ECL Select instead of ECL Advance
« Reply #3 on: October 31, 2012, 07:52:19 AM »
Hi, That´s true, ECL Select is a good replacement for ECL Advance. It has very high light output which gives a very high detection sensitivity and also allows highly diluted antibodies. The detection sensitivity is definitely comparable with ECL Advance but in general it requires slightly higher Ab concentrations, but it is still  very high dilutions compare to what other reagents requires.
This is my recommendation how to change from ECL Advance to ECL Select.
Use PVDF membrane for best sensitivity, as this types of membrane has higher protein binding capacity, but don´t forget to pre-wet in methanol before transfer. But nitrocellulose membrane works fine as well.
Use the same blocking as you normally use. However, the ECL Prime blocking (our former ECL Advance blocking) is a very good blocking as it is amixture of several blocking compounds.
Use 2-4 higher concentration of primary Ab compare to what you used for ECL Advance. typical dilutions are 1:3000-1:10000, depending on quality/affinity/protein levels. As an example 1:10000 works fine for more highly expressed proteins like GAPDH, ERK1/2.
wash 5 times 5 min
Dilute secondary Ab 1:50000-1:100000.
Wash 5-6 times 5 minutes.
So to summarise, use the same protocol/procedure as for ECL Advance but increase antibody concentrations 2-4 times.
I can really recommend you to try ECL Select. Good luck.