I have already reduced the concentration of primary antibody and incubation time with ECL. It worked but my 3 last blots look like that again. Any suggestions?
photo in the attachment
Thats because the substrate is getting used up quickly at the centre than at the periphery, due to either 1)lot of protein being present in the lanes, 2) lots of antibound bound to the protein. Just by increasing the dilution of your primary and secondary antibodies (you should be able to standardize it) will help you get rid of the "ghost bands" and also enable you to get sharp and crisp bands.
Kaushik is correct, your ghost bands are because of ECL substrate burnout. You have **too much** development occurring on your bands with the white center. This is a classic case of less is more for getting the perfect western. Use less primary and secondary (or as Kaushik said, increase your dilutions) and you should be able to reduce ghost banding.
Regarding the smears, do you reuse your running/transfer buffers? If so, try with fresh buffer and see if that affects the smearing. Also, do you reboil your samples before loading? Try to do so and load before they reach room temperature again.
Thanks a lot Jordan! Yes, I reuse my running and transfer buffer :) The first one is commercially availalbe and they say it can be used up to 10 times but I use maximum up to 5, otherwise my electrophoresis would last ages. And yes, I will reduce the dilution of my primary antibody as a first one. Thank you all for the answears!!!
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