Hi everyone，
1. Our lab have two chemiluminescent substrate from Thermo Scientific (moderate) and Millipore (very sensitive). For the same blot, these two substrates get different results.
2. Dispite of the same substrate, different exposure time may get different results.
see attachment for detail.
I am so puzzled about these two problems.
Note: I often add substrate mixture onto the blot and incubate for 30 seconds at room temperature.
Does anyone can give me some advice?
Sincerely
zhiyuan
Different exposure times will yield different results because chemiluminescence is based on an enzymatic reaction. The intensity of your signal is lost over time, this is why you can not recapture your WB again after, lets say, one hour of adding the substrate solution.
So in your case, the millipore substrate was more potent and thats why you get the high density of the bands. In your lower example, you started (probably) with the short exposure time and got signal x. However, time passed and this lowered your signal by z (notice that the decrease in signal is NOT linear!). So when you then exposed the blot for a longer period, your signal did not increase but rather decreased. Why? Because your exposure time was not enough to compensate for the loss in signal.
What do you do about that? You can't do a lot about it, thats why WB is not a quantative technique. If you want it to be quantative then you should go over to the infra-red detection systems (Li-Cor imaging systems) which is WB on a whole different level.
I hope your question is answered.
Different exposure times will yield different results because chemiluminescence is based on an enzymatic reaction. The intensity of your signal is lost over time, this is why you can not recapture your WB again after, lets say, one hour of adding the substrate solution.
So in your case, the millipore substrate was more potent and thats why you get the high density of the bands. In your lower example, you started (probably) with the short exposure time and got signal x. However, time passed and this lowered your signal by z (notice that the decrease in signal is NOT linear!). So when you then exposed the blot for a longer period, your signal did not increase but rather decreased. Why? Because your exposure time was not enough to compensate for the loss in signal.
What do you do about that? You can't do a lot about it, thats why WB is not a quantative technique. If you want it to be quantative then you should go over to the infra-red detection systems (Li-Cor imaging systems) which is WB on a whole different level.
I hope your question is answered.
And I have met the opposite phenomenon：The signal began decreasing but got steady for a longer time exposure. Why?
Hi Zhiguan, about the second case (protein Y)...it is no usual that after a longer exposure the signal decrease... This two images are taken from the same WB in two different times? same membrane with the same primary and secondary antibody?
Or 2 membrane: 1 membrane short time and the other membrane long time?
About the first case, I had a problem like you and in my case with the very sensitive kit my bands were saturated in the first time of exposure out of the linear range ( I couldn't see real differences), FInally I mixed 1:1:1 Component A, component B and Water.
About your case I don't know the details...but It can be possible that in the second image with millipore kit you aren't in the linear range?
Hi Erica,
For protein Y, the two images were from the same membrane and exposed to different time of chemiluminescent subtrate. And I have get the opposite results, that is, shorter exposure time decrease the protein level and longer time gets steady.
For sensitive kit, do you find some problems for mixing the substrates(A and B) with water? I just want to try this method and I am wondering whether it will produce different or wired results compared with the substrates only.
Hi Zhiyuan,
I had no problems mixing the substrates...DIluting the substrates decreases a little bit the signal (you have more time until the band saturation) . I only do that depending on my protein, if it is saturated with short exposure times. Another way that I use is adding the substrate mixture when you have the membrane in the detection instrument and in this moments starts to exposure the membrane. Also you can obtain more images until the saturation point.
From a company they told us that the signal is in linear range only 2 to 7 minutes after the addition of the substrate. Oversaturation may also play a role. The general opinion is that the films saturate much faster and possess short dynamic range, but the imaging systems are claimed to be much more accurate for a longer exposure. Hope that it helps a bit.
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