Loading control?

[Labnovice]

HI,

Here's one another beginner question. I am about to do my first WB tomorrow and am wondering about loading controls.
You run ur protein samples on a gel with a ladder... and then do western using primary antibodies against ur protein of interest
and then secondary antibody. Where does the loading control come in picture?
I bet its a silly question but I was thinking you load duplicate wells and
slice ur gel after it has run, and probe with antibodies against target and say actin...simultaneously.

Thanks for any suggestions.

[kmunson779]

Running duplicate lanes sort of defeats the purpose of a loading control -- you want to show that there are comparable amounts of protein in the actual lanes you loaded.

My labmates often choose a loading control protein that's a different size than the POI, and cut the membrane horizontally after transfer, probing each part with the appropriate primary and secondary antibodies and exposing them together.

Alternately, you could probe for your protein of interest, then strip the membrane and reprobe with your loading control antibody.