Hello Maafi Islam

I feel it is not the shadow but a fainter second band. It is possible that the protein bands may appear as doublets (two bands close together), which may indicate the presence of different isoforms.

Best

Malcolm Nobre Thank you for your reply.

The band on well 7 ( no shadow) is the same protein loaded in the same amount; however, a previous graduate student prepared that sample. It is a membrane phosphoprotein. The membrane was extracted by the Mem-Per kit from Thermofisher. I am following the same protocol. But I don't know why the previous student didn't get any dual bands and I am getting it.

It's a double-band with minor ~5 kd modifications of the same protein. It usually does not pose an issue for the presentation or quantification of the data. As the protein preparation seems to affect this, some protein fragments likely fall off during sample prep, producing the second band. Suppose you want to demonstrate your target as the double band. In that case, you can try to run the gel longer, allowing smaller sizes to run through to increase the separation between the bands. Still, overall, the data you are presenting is of perfectly publishable quality unless there is some significance for having larger and smaller bands.

Anton Lennikov Thank you very much!