Hi everyone,
a very weird phenomeon is occurring in my lab. In order to detect some proteins that are expressed at low level I am using an ECL kit (for high level-expressed proteins I normally use DAB). ECL detection goes fine and post-detection PVDF membranes are kept in the dark at 4 °C. The very strange thing is that two or three days later protein-specific dark bands become visible, as if WB had been revealed by DAB-based detection. I checked for every possible DAB contamination and used new plasticware for incubations and blot manipulation, however I am still experiencing this "problem".
How could that be possible?
Thanks everybody for any help.
Stefano Falone
University of L'Aquila
Can u share the image u got . It will help me in identifying the problem. Which ECL detection Kit and DAB kit you are using to develop the membrane
Metal Enhanced DAB Substrate Kit (ThermoFisher Scientific, cat. 34065)
LiteAblot EXTEND Long Lasting Chemiluminescent substrate Euroclone, cat. EMP013200)
Here You can find attached the two images of the same membrane, which was first developed by chemiluminescent reaction and then (spontaneously) by seemengly DAB-mediated precipitation.
Thanks in advance
Stefano
But this is not affecting your results. You can check your ECL kit expiry...
The ECL kit is not expired. I am still trying to figure out what is going on.
Thanks anyway
Stefano
I am wondering if what you are experiencing is protein oxidation with time causing the discoloration that you are observing.
It seems to be the band that is reacting with your primary antibody... correct?
You may be amplifying the amount of protein associated with your target band by applying a primary and then a secondary that has a 44 kDa protein attached to it (HRP). Essentially, you are adding quite a bit of protein on top of your target band associated with the PVDF. HRP has iron associated with it, when this oxidizes it will discolor.
That makes sense. I will try to avoid oxidation by degassing the TBS and sealing the reaction environment. Thank You very much for Your precious help.
Stefano
I have seen this happen as well when analyzing highly overexpressed proteins from transfected cells. I assumed it was some byproduct of the HRP reaction. The oxidation of the iron is a great explanation. It's never really been an issue because I only reuse blots on rare cases when I want to strip and reprobe. If this is why you store your blots after developing, perhaps you could strip them immediately to get rid of the secondary antibody. Then you could store them in TBS without the color developing.
Hi
It's a routine phenomenon I observe when I develop my blots using ECL.
If you keep the blots exposed to light, the bands will appear sooner than 2 days.
My guess is that the ECL bound to detection antibody somehow forms a visible complex when the blot is stored or exposed to light.
Also, the process and duration of development of these visible bands is directly proportional to the chemiluminescence signal, i.e., more intense is the signal, faster is the band visualization process, and thus, shorter is the duration of the process.
Even I couldn't find a satisfactory answer for this phenomenon.
Shridhar,
As I said in a previous post, the likely suspect is iron oxidation giving a rust colored appearance to the band. It makes sense that you see a correlation with stronger ELC, which would indicate more deposition of the secondary bound to HRP.
If you were to store an HRP conjugate at 4 C for a while, you will start to see more accumulation of brown particles in the tube with time. This is due to oxidation of the enzymes iron center leading to a brownish rust coloration. This is probably happening on the western membrane, but at a quicker rate.
Stefano, I agree Michael Shapiro that if you strip your blots that this issue will be resolved. I typically strip by blots using 2M Glycine at pH 2.3 with great results.
Western blot Mild stripping
Buffer, 1 liter
150 g glycine (2M)
1 g SDS (optional) (0.1%)
10 ml Tween 20 (optional)
Adjust pH to 2.3
Bring volume up to 1 L with ultrapure water.
Membrane incubation
Use a volume that will cover the membrane.
Incubate at room temperature for 5-10 minutes (longer if desired).
Discard buffer.
Repeat stripping with fresh buffer.
Discard buffer.
5 minutes TBST
5 minutes TBST
Ready for blocking/probing stage.
Thank you all, guys. Researchgate and its members have been of great help in learning something new.
Have a nice day.
Stefano
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