Target protein band is very weak, why there is a strong signal at the top of a gel. What can be a reason? 

    Some proteins may aggregate if the sample is heated in presence of SDS at 100°C for 10 minutes, as in this example. Aggregated target protein will therefore stay at the top of a gel and a signal from actual target will be weak or not present at all following development of a Western blot. 

    An example of such protein is plant H+ATPase. 



       Protein aggregates at a top of a gel

     Samples: Arabidopsis thaliana total and plasma membrane protein.
    Target MW: 90-95 kDa (depending upon gel system used)
     
    To solve this issue: sample should be denatured at a lower temperature of 70°C.


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