This is more a question than an answer.

But wouldn't it be the antibodies that limit the detection? So the 100 pg are the sensitivity in a very best case scenario.

I have observed different levels of signal when using different sets of antibodies on the very same lysate.

Hi Jennifer,

I've always used ECL rather than colorimetric or fluorescent detection for developing Westerns, but people in our Department who have used both say that ECL is more sensitive.

It also depends what imaging method you are using. If you're using a CCD imager (digital camera based system) then you can buy ECL reagents with extremely high sensitivity from various suppliers; much enhanced over using film. If you're using film, your detection won't be as sensitive with the standard ECL reagents (or even ECL plus).

One final point... I assume you tried a range of primary antibody concentrations to attempt to optimise the detection? If you're seeing a faint band then doubling the primary concentration will usually make a lot of difference, in my experience.

Hope that is helpful, and good luck!

Mark

Hi Jennifer,

Previous comments have provided some information about working with purified proteins but what I understand that you have cell lysis, and only a very small part of your extract contains your protein that you want to detect. What is the concentration of this protein may be difficult to measure and time consuming. I can give you a number of suggestions on how you can detect very small amounts of proteins.

A- You can amplifying the signal of your protein by immunoprecipitate your protein then continue with WB.

B- If you load the extract directly. Then you can use a number of enhancements:

1 - Use the ECL Select but wash the membrane five times before detection steps. ECL Plus can provide much higher background which impairs protein detection. Use CCD camera that is much more sensitive than film.

2 - The detection can be amplified 100 times if you use Signal Booster (from beacle, Inc. Japan).

3 - You can use the ECL Plex. Cy5 conjugated antibody ..

Good Luck

HI Jennifer,

the detection limit of a protein depends on two facts:

1. The content of your specific protein. It can goes down to 10 pg/lane.

2. The affinity of your antibody. The higher the affinity is the lower will be the detection limit. This is much more important than the protein content!!!!

So, if you can, try several specific antibodies.

If you are comparing an protein stain (the best is a gold stain) with an immuno-stain, you will find there huge differences.

here is really good paper on own made ECL reagents:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271654/

als try TMB from sigma with HRP instead of BCIP/NBT. In my hands it is often much more sensitive. 50 pg AP is visible wit it. of course more (or sometimes less) enzyme is at the end bound to your blot membrane. But usually the signal is amplified with the secondary antibody. Like 15 pg of protein is there, then maybe 5 pg of primary mouse antibody is binding (if the protein has 50 kD), to that then maybe 50 pg of seconday polyclonal anti-mouse-AP (if there are 10 epitopes detected on the primary mab by the polyclonal secondary) is binding.

you can further amplify the signal by increasing this amplification. e.g. instead of using anti-mouse-AP in our example, you could use anti-Mouse-Biotin, again maybe 50 pg might bind. followed by streptavidin-AP. If now about 10 biotin molecules are on the secondary antibody, 500 pg strep-AP will bind to the initially 15 pg protein in the band. You can get a tenfold increase in the signal at the end.

However specifity will be worse as any reagent has off-target binding. but it can work.

There are also new methods which ampfly the signal by placing additional dye molecules on the spot. but those are based on fluorescence usually instead of chemilumiescene. The most often used ones are based on tyramide:

https://www.thermofisher.com/de/de/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/tyramide-signal-amplification-technology.html

you can read about this amplification by indirect detection methods here, even with a video :-p :

https://www.thermofisher.com/de/de/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/introduction-secondary-antibodies.html

does anyone increase the temperature of incubation with the substrate? because AP (and HRP) have higher than room temp activity maxima.

like 50 degrees for AP:

https://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/alkaline-phosphatase.html

oh. I forgot to upload the titation with HRP (from biolegend, strep-AP) and TMB (sigma, ready to use for membranes). looks a but ugly, but one can see a clear nice dot at 50 pg.