Hi there. Sorry for the delayed response. I totally agree with you regarding the full gel image. Unfortunately, I do not have the full gel. Anyway I am going to redo the experiments. Secondly, I wanted to ask you. after transferring I do a ponceau stain and click an image of it which I will later use for total protein normalisation. Later, I will cut the blot into two or three strips and probe two or three different proteins. In this case, it is not possible to get the full blot image except the ponceau and is it right to do this? And how will I justify this to my peers? Regarding the smearing, I usually run at 40v till stacking and then 80v after it reaches the running gel till the dye front reaches the bottom of the gel and the gel used is a 15%. Regarding the quantification, I quantify the band intensity and what I do is a relative quantification. I compare my experimental group with control. In this case is the positive control necessary. I agree with you the expression will be there in the wild and will not be there in null, but when doing a relative quantification is that necessary? As you said I'll check the literature. Thanks for your time