I did western blot, oaded 150 ug per well, transfer was also well, but coudnot see any bands in chemidoc, used ECL in presence of light.
Hello Rashmi mishra Subodh
The substrate working solution is light sensitive. So preferably avoid exposure to intense light. Short-term exposure to laboratory lighting should not harm the substrate.
May be the HRP you are using, or the substrate has low activity. You should use the substrate working solution within 30 mins of mixing.
You should also ensure that the buffers like transfer buffer, TBST, running buffer as well as ECL are new and non-contaminated. If buffers are contaminated, especially with sodium azide it can inactivate HRP. Another reason could be the use of blocking agent like non-fat dry milk which could mask the antigen. So, decrease the amount of milk as well as the blocking time. You can use BSA instead. Also increase the antibody concentration and reduce the total protein loading concentration to 30-40ug per well.
Best.
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