ECL detection limits

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Guy Sovak
Guy Sovak's picture
ECL detection limits

Dear All,
Dose any one knows what is the detection limits of ECL?
And what about its linearity?

Mulli
Mulli's picture
It really depends on the

It really depends on the antigen and the quality of your primary antibody.

The accessibility of the affinity tags varies with the structure of the individual protein. It also depends on where the tag is attached. Therefore, the detection limit varies from one tagged protein to another because the tag on some proteins is poorly accessible to the antibody.

Long story short. The lowest detection limit could be anywhere from .1-500ng.

Guy Sovak
Guy Sovak's picture
Thanks for the answer

Thanks for the answer
and welcome to our board
Guy

Edward Dougherty
Edward Dougherty's picture
For information on the

For information on the potential linearity of ECL, see:

MOLECULAR PHARMACOLOGY 52:202-211 (1997).
Determination of Aryl Hydrocarbon Receptor Nuclear Translocator Protein Concentration and Subcellular Localization in Hepatic and Nonhepatic Cell Culture Lines: Development of Quantitative Western Blotting Protocols for Calculation of Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Receptor Nuclear Translocator Protein in Total Cell Lysates

Jennifer L. Holmes and Richard S. Pollenz

http://molpharm.aspetjournals.org/cgi/content/full/52/2/202

In particular, look at figure 1, which shows linearity of ECL from 12.5 to 250 pg of protein. As previously mentioned, though, the efficacy of your antibody and cellular concentration of protein will impact the linear range; however, each protein/antibody should be linear over a given range.

http://molpharm.aspetjournals.org/cgi/content/full/52/2/202

mchinmoyee
mchinmoyee's picture
To know about right quantity

To know about right quantity of protein or antibody dilution to be taken, one can do a dot blot or run a SDS-PAGE with different concentration of protein sample with increasing concentration (e.g; 1,2.5,5,10,15,25,50ug and more) and when you detect it by chemiluminescence the quantitation will give you linear curve(almost) and this will help you decide how much protein is to be loaded to get in adetection range and if samples are limited you can play with of primary or secondary antibody concentration or a much better substrate like advance ECL.
Linearity also tells about the range of over saturation which should be avoided in any case.
This graph shows an example of it but has used different substrate.

manyquestions
manyquestions's picture
Hi, I have a very stupid

Hi, I have a very stupid question regarding the dilution.

Does the dilution that I prepare have to be strictly linear?

like everything has to be separated in steps of 2.5?

1, 2.5, 5, 7.5, 10, 12.5....etc

if you don't have these fixed steps, and you have numbers like:

1, 2.5, 5, 10, 15, 25 (that are not increasing linearly ) would that still give you a linear plot?

thank you?

mchinmoyee wrote:

To know about right quantity of protein or antibody dilution to be taken, one can do a dot blot or run a SDS-PAGE with different concentration of protein sample with increasing concentration (e.g; 1,2.5,5,10,15,25,50ug and more) and when you detect it by chemiluminescence the quantitation will give you linear curve(almost) and this will help you decide how much protein is to be loaded to get in adetection range and if samples are limited you can play with of primary or secondary antibody concentration or a much better substrate like advance ECL.
Linearity also tells about the range of over saturation which should be avoided in any case.
This graph shows an example of it but has used different substrate.
Jason King
Jason King's picture
It doesn't matter whether you

It doesn't matter whether you have equal increases at each concentration. Similarly, if you're doing a time course reaction where you measure ODs on, say a 96 well plate over 24 hours, you don't NEED to book the plate reader for exactly on the hour.....but try telling the undergrads this! ; )

manyquestions wrote:

Hi, I have a very stupid question regarding the dilution.

Does the dilution that I prepare have to be strictly linear?

like everything has to be separated in steps of 2.5?

1, 2.5, 5, 7.5, 10, 12.5....etc

if you don't have these fixed steps, and you have numbers like:

1, 2.5, 5, 10, 15, 25 (that are not increasing linearly ) would that still give you a linear plot?

thank you?

mchinmoyee wrote:

To know about right quantity of protein or antibody dilution to be taken, one can do a dot blot or run a SDS-PAGE with different concentration of protein sample with increasing concentration (e.g; 1,2.5,5,10,15,25,50ug and more) and when you detect it by chemiluminescence the quantitation will give you linear curve(almost) and this will help you decide how much protein is to be loaded to get in adetection range and if samples are limited you can play with of primary or secondary antibody concentration or a much better substrate like advance ECL.
Linearity also tells about the range of over saturation which should be avoided in any case.
This graph shows an example of it but has used different substrate.
mchinmoyee
mchinmoyee's picture
Ya , it will work , only the

Ya , it will work , only the slope will change most probably.

manyquestions wrote:

Hi, I have a very stupid question regarding the dilution.

Does the dilution that I prepare have to be strictly linear?

like everything has to be separated in steps of 2.5?

1, 2.5, 5, 7.5, 10, 12.5....etc

if you don't have these fixed steps, and you have numbers like:

1, 2.5, 5, 10, 15, 25 (that are not increasing linearly ) would that still give you a linear plot?

thank you?

mchinmoyee wrote:

To know about right quantity of protein or antibody dilution to be taken, one can do a dot blot or run a SDS-PAGE with different concentration of protein sample with increasing concentration (e.g; 1,2.5,5,10,15,25,50ug and more) and when you detect it by chemiluminescence the quantitation will give you linear curve(almost) and this will help you decide how much protein is to be loaded to get in adetection range and if samples are limited you can play with of primary or secondary antibody concentration or a much better substrate like advance ECL.
Linearity also tells about the range of over saturation which should be avoided in any case.
This graph shows an example of it but has used different substrate.
Shubhangi
Shubhangi's picture
Hi

Hi
it will not be a problem if your concentrations are not linear. In case it is not giving linear curve, use some formula to transform your data in linear curve. say for example convert your concentrations into log values and plot a graph. similarly one can transform OD/ RLU/RFU values to log or equivalent terms.
 

Zhu Milk
Zhu Milk's picture
ECL detection limits are based on your ecls

Yes. The family of ECL substrates is a series of premixed, ready-to-use chemiluminescent reagents for the detection of HRP-based Westerns. SuperLumia ECL Chemiluminescent HRP Substrate Reagent is specially optimized for picogram amounts of antigen detection on immunoblots, with pouring directly onto the blot membrane without worrying about pipetting error. The substrate's intense signal output and duration enables detection of picogram amounts of antigen with photographic or other imaging methods perfectly well.