ECL detection limits

guy

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Nov 16, 2015
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#1
Dear All,
Dose any one knows what is the detection limits of ECL?
And what about its linearity?
 

Mulli

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Nov 16, 2015
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#2
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.
 
Nov 16, 2015
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#4
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

New member
Nov 16, 2015
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#5
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.
 
Nov 16, 2015
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#6
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 said:
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.
 

parvoman

New member
Nov 16, 2015
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#7
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 said:
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 said:
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.
 
Nov 16, 2015
32
0
0
2
#8
Ya , it will work , only the slope will change most probably.

manyquestions said:
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 said:
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

New member
Nov 16, 2015
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#9

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.



 

biomind

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Jan 25, 2017
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#10

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.