But what you need to do?

If you need to run a 2D gel you need ampholytes;one direction of the two in a 2D gel is separation by pH/pI and ampholytes are thus essential.

Thank you franco sir.  So its clear that ampholytes are very essential for ph gradient separation of protein in IEF.

Is any substitute of ampholytes and can they be prepared in lab?

Look similar questin in research gate

https://www.researchgate.net/post/How_can_I_prepare_ampholytes_for_2d_gel_electrophoresis

Thanks a lot Franco sir for your nice suggestions. It will be more helpful for me.😊😊😊

Yes, It's essential for separation pH/pI in 2-DE gel.