When analyzing proteins, how should we prepare run running buff

Greetings.
I have a question about protein-protein kintics.
I use Dojindo amine coupling kit during protein analysis.

In there protocol,
1) equillibrium with PBS
2) WSC/NHS (solute in activation buffer)
3) PBS washing
4) Protein G (in activation buffer)
5) PBS washing
6) Ig G (in activation buffer)

but in that kit, activation buffer is too low amount, so we substituted 0.1M MES(pH 7.4) for activation buffer. (because it is sufficient but other component is remain every time) It works.

However, in this procedure, which substituted 0.1M MES for activation buffer, bulk effect occured.

In this case, is it adequate to make the running buffer MES buffer instead of PBS?

Dear KS,

You can substitute MES for PBS. With amine coupling it is imperative not to use any buffer that has free amines.

The buffer strength of the running buffer should be normal (10-20 mM MES, 150 mM NaCl).
Keep the buffer strength of the ligand buffer low (10-20 mM MES) during the immobilization and adjust the pH 0.5 units below the pI of the ligand.

The differences in buffer composition en strength causes the bulk differences between the steps of the immobilization. This is perfectly normal.

Please look at the figure at
www.sprpages.nl/immobilization/immobilization-procedures/amine

Kind regards
Arnoud Marquart

Dear Arnoud.
Thank you for your answer.

We use 0.1M MES buffer(pH 4.7) why Dojindo said 'you can substitute activation buffer to pH 4.5 buffer', and we have Thermo fisher MES(0.1M pH 4.7). we use this buffer when run amine coupling.