Maybe you have some ideas about where this backround is coming from? I am not sure if this comes from blotting, blocking or if this is dirt? Thank you very much in advance!
Hi Fabian,
as far as i understand from the pictures your membrane was either touched or blocking step was not successful or they are dirt. First of all, please be sure that your membrane was packed, untouched, and clean before you put it under your gel. You have to take it on the edge with the help of tweezers without touching the membrane. Bubbles should be removed after you put filter paper on gel. Be sure all your solutions are dust-free and fresh. Blocking should happen at room temperature for at least1 h.
Good luck!
following can be the reasons for this type of blot:
Improper blocking (Use fat free skimmed milk or BSA as blocking reagent)
Inadequate washing (use 1X TBST with 0.1% tween-20 for washing)
Touching the membrane directly
Mishandling of membrane due to which it could have came in contact with unclean surface
You can also look out the protocol for western blotting provided by Biorad, Sigma or Abcam to find out your mistake.
Hello Fabian Recker
You could follow the steps below and hopefully it should work.
1. Prepare fresh buffers. The presence of bacteria or other contaminants will severely affect blotting.
2. You should limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and increase nonspecific background.
3. Ensure the equipment used is clean. The presence of contaminants on dirty equipment may result in blotchy or speckled patterns. Use clean incubation trays or dishes. A rinse with methanol followed by water will help dissolve residual dried dyes from previous uses. Clean the transfer devices and dusty consumables if using a wet transfer method, as they can introduce speckles. Clean the imager surface with ethanol to remove dust, lint, and residue before capturing the image.
4. Poor handling of the membrane could be one of the cause. Membranes when poorly hydrated before transfer can cause this type of problem. PVDF requires methanol for proper hydration. If the membrane dries out during the procedure a high background may occur. Old membranes can also severely affect the quality of your blots. Use clean forceps to handle the membrane and avoid directly touching the membrane.
5. Ensure good coverage of the whole blot during all incubation steps.
6. Ensure consistent agitation during every incubation step.
7. Even exposure time could be a cause. So, try reducing the exposure time.
Best.
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