Loading Controls

[kiki06]

Hey!, i need some ideas for loading controls...

what do you guys use for loading controls in western blot?

beta-actin?
tubulin?

what is the best to use?

[amicia]

Hello Kiki06,
the answer to your question is depend:
you have to choice which type of control works better. For exemple, in human studies (cell cultures and tissue) I used actin, beacuse is really well express in many cellular compartiments (cytosol, but also mitochondia and for somebody also in nuclei), but also gamma-tubulin generally works very well, but in my knowledge this last one is used only for cytosol protein.
Moreover, if you're evaluating the phosphorilation state of some proteins, I suggest you to use antibody against the phosphorilated state and the non-phosphorilated state, in order to compare the difference between the different treatments in phosphorilated state (of course the amount of no-phopho has to be the same). hope to have help you.
regards

[kiki06]

thank you Amicia!


I think I will need to check if I can use gamma-tubulin...

[shalmoli]

I think actin will be a good choice

[agador]

I've always used GAPDH as a loading control the signal is quite robust but then again it depends on your experiment... we dont use actin in our lab as it is downstream of our pathway of interest.