Hi!I have recently encountered problems with the Western blot analysis. I get blots with high background(all black!) and some white spots. After stripping and using the same antibody, I had also black background and white spots in different places than before.Blocking and antibodies are the same.(This used to work fine).Do you have any idea?
Did you change batches of any of your reagents? Sometimes, even if you buy the same reagent from the same vendor, batch-to-batch variation can mess up your assay.
from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
N Am J Med Sci. 2012 Sep; 4(9): 429–434.
doi: 10.4103/1947-2714.100998
PMCID: PMC3456489
Western Blot: Technique, Theory, and Trouble Shooting
Tahrin Mahmood and Ping-Chang Yang
"High background is often caused by too high concentration of the antibody, which can bind to PVDF membranes. Another problem could be the buffers, which may be too old. Increasing the washing time can also help to decrease the background. Additionally, too high of an exposure can also lead to this problem. Therefore, it is advisable to check different exposure times to achieve an optimum time."
Could be a lot of things:
ECL substrate still good? Too much ECL? Blocked for sufficient time? How do you block? Blocked at all? First and/or second antibody dilution too low? Exposure time too long? Old buffers? Tween added to buffers? What do you take for stripping? Could the stripping solution have affected you membrane quality? Just some ideas...
Did your membrane dry out? If so then you will obtain black background. If there is too much ECL you will get white spots and black background. Biorad ECL much more sensitive than Pierce.
Your full protocol with primary and secondary antibody dilutions would be helpful. I'd say that one or both antibodies are at too high a concentration. That can cause precipitation of the luminol on your bands, preventing the background chemiluminescence from getting to your film and producing white bands. The uneven background can also result from too much residual ECL remaining on parts of the blot during exposure. Without an exact protocol and technique, though, we can only throw out lots of ideas.
Thank you all for your help!The antibodies dilution or blocking conditions hadn't changed, I use Biorad ECL and my membrane hadn't dried out. These blots kept appearing in this way so I checked the buffers we all use in the lab. It seems that the pH of TBS had changed due to changes in room temperature. I hope this has been the problem!
Hi,
I had high background in my westrnblot before , and your result showed bands, it's good thing, we usually first see "black picture", then try to get "clear picture".
I have 3 new advise :
1.Diluted your 1st and 2nd antibody in your blocking buffer, especially when you use the unpurifyed 1st antibody.
2.Diluted the 2nd antibody further, even to 1:100,000, while it depened on your antibody type.
3. Try to use CCD camera to detected your signal, if it possible.
Hope it helps!
DUERNA
Hi Penelope,
I think the problems arise through the use of much too much secondary abs, I would suggest to dilute the 2° abs much higher (normally the dilution depend on the substrate but with very sensitive substrates you can go up to 1: 100 000 or even 1: 500 000). The "white" bands are the areas where the the 2° abs bind to the primary abs and in this region the substates was already completely transformed to product (so no light emission) when you start your detection.
Best,
Carsten
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