Hello Adeseye,

With CN/DAB staining, depending on the amount of the protein and resulting intensity of the band, it is possible to see bands on the other side. If you use 0.2 um membrane, perhaps the bands will not be as visible as they are on the 0.45 um membrane.

Protein with higher concentration, there might be a possibility to appear protein bands on both sides. But using 0.45um NCP membrane with protein concentration of 3-4 mg/ml using DAB as a stain, we never got the bands on both the side. I think the concentration of protein is much high, you need to dilute and reload it, you will get the fine results.

This Sounds more like a Transfer issue than amount. I have loaded from 5ug to 50ug total protein in a well and never saw a band on the other side. I am guessing you are doing high voltage transfer. Also the pore size of the membrane (0.45) could be a probable cause. If the intensity of the band is really high then you could try loading less protein, however that may not solve the problem. Try doing lower voltage and longer time transfer. By keeping the lower voltage you are giving the protein more time to bind with the membrane and there are less chances of them to blast through.

Hope this help!

Thanks everyone for all the suggestions. The bands still appear on both sides of the membrane after reducing the protein concentration and also the time for the electrotransfer. The next option am going to try out is using a membrane with a lower pore size. Hopefully that works. Thanks a lot.

Your target is quite small so it can happen that the proteins blot through your membrane and even appear on your Whatman paper. Reducing pore size and the strength of electric current might help. If not, try to blot without electricity. Dry blotting is an old method that is still used for southern blots and works very well with small proteins. Without the electrical force the possibility of "over blotting" is very small. Another advantage is that you do not need additional buffers. The sandwich should look like this:

top                                  a book or the like

                                  5 layers Whatman paper

                                    activated membrane

                                                  gel

bottom                          clean plastic foil

Do not use too much pressure (a too heavy item on top)! The capillary force does the trick. Blotting time depends on protein size. From our  experience (we blot our large film-backed 2D gels this way, with the Beo Dry Blotter) an over night blot works very well.

I hope I could help.

Hi Kristin, thank you for your detailed explanation. Using a membrane with a smaller pore size (0.2 um) helped solve this problem. Was able to get the protein bands on one side of the gel. The method you suggested is really advantageous (cost saving), and I might try it out in my subsequent western blot experiments. Thanks