Hello Haley Luu

Loading control is used to show whether protein loading variation has occurred and may account for observed variations in the target band(s). When used correctly, loading control ensures that proteins are quantitated appropriately despite subtle differences in loading amounts across all lanes of the Western blot.

Some points to bear in mind while using the loading control in Western Blot.

Ensure that your protein of interest (alpha-SMA) and loading control have different molecular weights because this will allow you to easily distinguish bands representing your protein of interest from those representing your housekeeping protein (loading control).

Select your loading control in such a way that it is highly expressed in your sample and its level remains unchanged throughout an experiment. Don’t use a loading control that you’ve found to vary in expression level during your experimental treatment.

Probing with a loading control can be carried out alongside the target antibody by cutting the membrane before antibody incubation, or after the blot has been stripped of previously bound antibodies.

You should always carry out titration experiments on your samples to determine the concentration of antibody against your housekeeping protein (loading control) and blot exposure time that gives you optimum signal-to-background ratio without signal saturation. This is because housekeeping proteins are often highly expressed, thus making it easy for the signal from the housekeeping protein detection to become saturated and affect normalization.

So, you need to change your loading control. Choose a loading control that is in a similar MW range, but not the same MW as the target protein. This ensures that both target and control bands may be easily distinguished on the blot.

You can used GAPDH with MW 37, or beta tubulin with MW 55, or alpha tubulin with MW 55.

Hope this is helpful.

Good Luck.

I agree with Malcolm..use GAPDH..

Try changing to another control such as GAPDH, Tubulin, α-Actinin.

https://www.cellsignal.com/applications/western-blot-immunoblot/loading-control-antibodies

https://www.bio-rad-antibodies.com/western-blot-loading-controls-antibodies.html