Dear Sanjay,

the constitutents of a 2D rehydration buffer are having different functions during the isoelectric focusing. Most important is the solubilization of the analytes, namely your proteins in your sample. By reconstitution of your lyophilized proteins in water only your proteins will maybe stick together form aggregates and you might face some serious precipitaion. There are tons of literature about this topic, please read some of them before continuing to waste your samples.

Best, Murat

Dear Sanjay,

You cant substitute rehydration buffer with anything. It contains urea, CHAP, DTT, Thiourea and Tris in particular amount and each has specific role for protein preparation for proper isoelectric focussing. Rehydration is must if you want protein spots after SDS-PAGE.

Thanks Murat and Anupama.