Hello Abhishek.
First of all, the noise did not exist simply during the chemiluminescence step. It could come anytime throughout the long process of western blot - starting from the gel casting to the image processing, i.e. from Day 1. But if you have troubleshooted, and found your error in the chemiluminescence step - good job for finding out the error! So here are the things that you may consider looking into:
1. Do you recycle your ECL? Not recommended. Use freshly made everytime.
2. Was the membrane immersed in ECL during incubation? Make sure the membrane is all fully covered.
3. Follow the manufacturer's advice - if they say 5 mins in ECL, do not exceed 5 mins. Do your imaging immediately after 5 mins. My ECL states 5 mins, I do my imaging at 4.45 mins-4.30mins, never more than 5 mins.
4. Do you share your imaging instrument with other researcher? Clean/Dry the surface before you use.
5. Did you do some warming up of the instrument before imaging? i.e. focusing/previewing standards to ensure that the instrument is imaging well.
6. Do you use plastic wrap to pull down your membrane during imaging? - The plastic might be dirty.
7. Remove excess ECL before imaging. Excess ECL will cause blotches on the blot.
Can try making up the antibodies in either BSA or Milk see if that makes a difference, i've found in the past certain secondaries actually react with BSA or Milk and give a very dirty signal. Could try also a different ECL reagent?
Can also try longer washing steps, I often have left blots much longer than the normal 3x5min washes after the primary and secondary antibody steps, even an extra hour and this produces really clean blots, some people I know even leave for a day washing.
1. Try different 2nd ab concentrations.If you already tried that:
2. Try the selfmade cheap stuff (Luminol + p-Coumaric acid + H2O2). It is usually good enough for detecting bands and gives you less background. I always got better results with the DIY System :) And it is a lot cheaper as well. If you need a protocol, let me know. I just use the expensive system for faint and difficult bands.
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