Yes, you can see differences in each lane with different amounts of protein. I am curious why you want to load different amounts?

Usually, on SDS PAGE, I will load different amounts in order to be able to see dilute contaminants, but on Western Blot, you would just end up seeing a difference in the size of the band of your protein of interest.

Also, what are you loading? I generally load 2-5ug of purified protein. If you're expecting a lot of different proteins, then 10-30ug might be ok, but with a purified protein, it seems like a lot.

Hi Lindsey Gallardo ,

I will be doing SDS-PAGE with cell total protein. I want to load different amount of protein because the target protein that I am looking for could be hard to detect. I thought it would be a good idea to load more so that it would be easier to detect. I am not sure if this is a way to approach or not, but it's just my thought.

Best,

Paul

Hi Víctor Hugo Urrutia-Baca

What do you mean by nonspecific band? I thought primary antibody would be specific enough to detect target protein in complete lysate?

Best,

Paul.

Sure, you can load different amounts of proteins in different lanes in one gel.

In case of purified proteins or precipitate from IP, it will not be a big problem. However, having a pool of proteins, the protein of interest might be a minor part, and overload of other proteins might squeeze neighboring the lanes if this contains much less protein, resulting in fat and tiny lanes.

Since you run WB, you might also be able to improve your results by changing blotting conditions (membrane type, transfer time, buffer system); some proteins needs longer transfer time, others run through the membrane. No straightforward answer what you should test, just try!

Best,

Olav