Hi,

Please find a good explanation below. The main reason is to differentiate the rate of migration while the proteins are stacking into a tight band in the wells, before they enter resolving gel for separation. The respective pH influences the charge of ions in the running buffer, and thus their migration when electric current is turned on.

https://www.researchgate.net/post/Why_do_we_use_Tris_solution_of_two_different_pH_during_preparation_of_SDS-PAGE

Hi,

Please find a good explanation below. The main reason is to differentiate the rate of migration while the proteins are stacking into a tight band in the wells, before they enter resolving gel for separation. The respective pH influences the charge of ions in the running buffer, and thus their migration when electric current is turned on.

https://www.researchgate.net/post/Why_do_we_use_Tris_solution_of_two_different_pH_during_preparation_of_SDS-PAGE

Hi Sonica,

Basically, buffers of different pH are used to maintain the charge of glycine used to prepare the buffer. Depending on the pH, glycine can exist in positive, negative or neutral charge and this affects the rate of the migration of protein in the stacking and resolving gels. A much detailed and clear explanation of how this works can be found in the link below. 

I hope this helps.

Stacking gel is so negative so samples will habe a nice negatively charged smack into the running gel from the stacking gel.