2D help

[raadhabio]

As I am new to the 2D gel electrophoresis technique, I have a number of questions. Basically, we perform the 2D gel electrophoresis with carrier ampholyte.

1) how to track the mobility of the protein in the IEF tube gel?
2) We used to run the IEF at
200 v for 2 hrs
500 v for 2 hrs
800 v for 16 hrs.
But in some references, it is given that once the protein reach their pI, there will not be any current flow and thus it gets stopped. Is it so?

3) I got a plain gel with a single streak. Does anyone help me in this regard?

[researcher123]

hi,
the only way of tracking the mobility of proteins in ief is by the bromophenol blue dye that must be there in your rehydration solution (in which you finally prepare your sample). However, it does not mean that when the dye has reacjed the acidic end (its negatively charged), IEF is complete; migration of proteins will still be taking place. Its just a way of keeping track in the begining for a few hours. Yes, once any protein reaches its pI, it will not migrate any more. This will continue till all have migrated to their respective pI. The volt hours taken depends upon your sample which you have to play around with a bit.