Loading control - (Apr/27/2006 )

Hi everybody,

I am realise some western blot for the moment and I would like to have the same loading between the different samples. After the transfert I realise a staining with Ponceau. The different bands look like the same. My question is: Can I be sure that the loading is the same if the ponceau's result is good? Or is it better to realise a bradford before my gel on my protein extract?

Thanks a lot for your consideration

Have a nice day

biofred

You should do both. Performing a Bradford should be matter-of-course for any work involving proteins. How can you possibly 'guess' how much you have. You're creating unnecessary optimisation steps otherwise.

And a second stain for the transferred proteins is essential. Even if you load perfectly that doesn't guarantee equal transfer. You need to show this as well.

Hi!
I agree. What I do is restain my transfer gel to make sure proteins are off of it. I also run a stained protein ladder with proteins of different size. See what doesn't transfer after one hour, two hours, etc if needed by peeling back just a small corner of your transfer membrane and carefully taking a look. Run a control transfer with same protein (if you have luxury of having some in stock).

I always run a matching coomassie gel to view along with my transfer. If transferring large, sticky, globular proteins, I will transfer overnight at 30V in cold room.

Best of Luck!

hi
i realize both. Bradford and ponceau staining. But best choice is to blot with a common Ab your all blots and expose membranes to the same film and reveal it. I done it for 50 samples as two ponceaus between membranes are not that accurate regarding the procedure of stain / decoloration...